Language selection

Search

Patent 2268265 Summary

Third-party information liability

Some of the information on this Web page has been provided by external sources. The Government of Canada is not responsible for the accuracy, reliability or currency of the information supplied by external sources. Users wishing to rely upon this information should consult directly with the source of the information. Content provided by external sources is not subject to official languages, privacy and accessibility requirements.

Claims and Abstract availability

Any discrepancies in the text and image of the Claims and Abstract are due to differing posting times. Text of the Claims and Abstract are posted:

  • At the time the application is open to public inspection;
  • At the time of issue of the patent (grant).
(12) Patent Application: (11) CA 2268265
(54) English Title: METHODS FOR OPTIMIZATION OF GENE THERAPY BY RECURSIVE SEQUENCE SHUFFLING AND SELECTION
(54) French Title: PROCEDES PERMETTANT L'OPTIMISATION DE LA THERAPIE GENIQUE GRACE A UN REARRANGEMENT ET UNE SELECTION RECURSIFS DE SEQUENCES
Status: Deemed Abandoned and Beyond the Period of Reinstatement - Pending Response to Notice of Disregarded Communication
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/00 (2006.01)
  • A61K 38/45 (2006.01)
  • A61K 48/00 (2006.01)
  • C07K 14/47 (2006.01)
  • C12N 7/00 (2006.01)
  • C12N 9/10 (2006.01)
  • C12N 15/10 (2006.01)
(72) Inventors :
  • STEMMER, WILLEM P. C. (United States of America)
  • VAN ES, HELMUTH H. G.
(73) Owners :
  • INTROGENE B.V.
  • MAXYGEN, INC.
(71) Applicants :
  • INTROGENE B.V.
  • MAXYGEN, INC. (United States of America)
(74) Agent: SMART & BIGGAR LP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 1997-09-26
(87) Open to Public Inspection: 1998-04-02
Availability of licence: N/A
Dedicated to the Public: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US1997/017302
(87) International Publication Number: WO 1998013485
(85) National Entry: 1999-03-24

(30) Application Priority Data:
Application No. Country/Territory Date
60/037,742 (United States of America) 1996-09-27

Abstracts

English Abstract


The invention provides methods of evolving nucleic acids for use in gene
therapy by recursive sequence recombination. Many of the methods evolve
vectors, both viral and nonviral, to have improved properties. For example,
vectors are evolved to have improved properties of viral titer, infectivity,
expression of a gene within a vector, tissue specificity, viral genome
capacity, episomal retention, lack of immunogenicity of the vectors or an
expression product thereof, site-specific integration, increased stability, or
capacity to confer cellular resistance to microorganism infection. The
invention further provides an isolated O6-methylguanine-DNA methyltransferase
(MGMT) enzyme.


French Abstract

L'invention concerne des procédés de mise au point d'acides nucléiques destinés à être utilisés dans une thérapie génique grâce à une recombinaison récursive de séquences. Beaucoup de ces procédés mettent au point des vecteurs, aussi bien viraux que non viraux, afin de présenter des propriétés perfectionnées. Par exemple, des vecteurs sont mis au point pour présenter des propriétés perfectionnées de titres viraux, d'infectivité, d'expression d'un gène dans un vecteur, de spécificité de tissu, de capacité de génome viral, de rétention épisomique, de manque d'immunogénicité des vecteurs ou d'un produit d'expression de ces vecteurs, d'intégration spécifique du milieu, de stabilité accrue, ou de capacité à conférer une résistance cellulaire à une infection d'un micro-organisme. L'invention concerne également un enzyme isolé O<6>-méthylguanine-ADN méthyltransférase (MGMT).

Claims

Note: Claims are shown in the official language in which they were submitted.


WHAT IS CLAIMED IS:
1. A method of evolving a drug transporter gene, comprising:
(1) recombinating at least first and second forms of the gene differing from
each
other in at least two nucleotides, to produce a library of recombinant genes;
(2) screening at least one recombinant gene from the library for conferring
improved or altered drug resistance;
(3) recombining, as necessary, at least one recombinant gene with a further
form
of the gene, the same or different from the first and second forms, to produce
a further library
of recombinant genes;
(4) screening, as appropriate, at least one further recombinant gene from the
further library for improved or altered drug resistance;
(5) repeating (3) and (4), as necessary, until the further recombinant gene
confers
a desired level of improved or altered drug resistance.
2. The method of claim 1, wherein more than one round of screening is
performed
between successive steps of recombining.
3. The method of claim 1 or 2, wherein the recombinant or further recombinant
genes are screened by exposing cells to a drug and selecting surviving cells,
the surviving
cells being enriched for recombinant or further recombinant genes having the
property of
conferring improved or altered drug resistance.
4. The method of claim 3, further comprising increasing the concentration of
the drug
between successive rounds of screening.
5. The method of anyone of claims 1 to 4, wherein the drug is a
chemotherapeutic
drug.
6. The method of claim 1 or 2, wherein the recombinant or further recombinant
genes
are screened by detecting efflux from cells of a substrate for a drug
transporter encoded by the
drug transporter gene or by the recombinant or further recombinant genes and
selecting the
cells containing low intracellular amounts of said substrate.
79

7. The method of claim 1 or 2, wherein the recombinant or further recombinant
genes
are screened by detecting influx into cells of a substrate for a drug
transporter encoded by the
drug transporter gene or by the recombinant or further recombinant genes and
selecting the
cells containing high intracellular amounts of said substrate.
8. The method of anyone of claims 3 to 7, wherein the cells are stem cells.
9. The method of anyone of claims 3 to 7, wherein the cells are kidney cells,
heart
cells, lung cells, liver cells, gastrointestinal or central nervous system
cells.
10. The method of any of the aforementioned claims, for use of the recombinant
or
further recombinant gene in gene therapy.
11. The method of claim 1 or 2, wherein at least one recombining step occurs
in vivo.
12. The method of claim 1 or 2, wherein at least one recombining step occurs
in vitro.
80

Description

Note: Descriptions are shown in the official language in which they were submitted.


CA 02268265 1999-03-24
WO 98/13485 PCT/US97/I7302
METHODS FOR OPTIMIZATION OF GENE THERAPY BY RECURSIVE
SEQUENCE SHUFFLING AND SELECTION
The present application is a Continuation-In-Part application ("CIP") of U.S.
patent application serial no. ("USSN") 08/721,824, filed September 27, 1996,
which was
converted to Provisional application serial no. 60/037,742, under 35 U.S.C. ~
111(b) and 37
C.F.R. ~ 1.53(b)(2); and a CIP of USSN 08/722,660, filed September 27, 1996.
Each of the
aforementioned applications is explicitly incorporated herein by reference in
their entirety and
for all purposes.
FIELD OF THE INVENTION
The present invention applies the field of molecular genetics to the
improvement of vectors and other nucleic acids for use in gene therapy.
Improvement is
achieved by recursive sequence recombination.
2 o BACKGROUND AND DESCRIPTION OF RELATED ART
Gene therapy is the introduction of a nucleic acid into cells of a patient to
express the nucleic acid for some therapeutic purpose. That is, the nucleic
acid is itself used
as a drug. For example, an appropriate gene can be delivered to a patient with
a recessive
inherited disease, such as cystic f brosis, to correct the genetic defect and
cure the disease
state. In other applications, delivery of genes encoding a toxin (e.g,
diphtheria toxin, ricin, tk}
can be used to kill cancer cells, and other genes can be specifically tailored
to kill infectious
organisms. Other applications include incorporation of regulatory sequences
near
endogenous genes. These different applications are directed to many different
target cells
with many modes of delivery (e.g., in vitro, ex vivo, in situ, intravenous,
and germline
3 o modification).
The power of gene therapy has led many large pharmaceutical manufacturers
and several smaller biotechnology companies to devote substantial financial
and technical
resources to developing gene therapy as a viable therapeutic approach to
treating human
1

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
diseases. Although simple in theory, gene therapy is not without technical
difficulties.
Development of any gene therapy requires identification of a cell type as a
target, means for
entry of DNA into those cells, means for expressing useful levels of gene
product over an
appropriate time period, and avoidance of host immune response to the gene
therapy agents.
The requirements for any particular application vary greatly and profoundly
influence the choice of vector to be developed and tested. Possible variables
in different
applications include the efficacy of gene transfer, the efficacy of gene
expression, the duration
of gene expression, the feasibility of repeat dosing, and the ability to
target appropriate cells
and avoid inappropriate cells. Confounding factors that may arise include the
inability of
virus or delivery vehicle to enter into or integrate into the chromosomes of
particular cells,
the shutdown of transcriptional promoters, the loss of input DNA, the
destruction of treated
cells, and the neutralization of input virus or gene product. All of these
factors depend on the
choice of viral vector or non-viral delivery system and on the ability of the
host to respond to
that virus or delivery system.
Most of the components currently available for constructing gene therapy
vectors were not evolved or developed for gene therapy, and thus may have many
undesirable
features and may lack efficacy in the desired gene therapy application. For
example, most
eukaryotic viruses have evolved to optimize virulence and viral reproduction,
and most non-
viral DNA delivery systems were designed to be used for experimental
transfection in
2 0 laboratory conditions, not for administration to humans.
Solutions to the above difficulties and inefficiencies are needed before gene
therapy becomes effective for routine treatment of significant numbers of
patients with
common diseases. The present invention fulfills this and other needs by
providing inter alia
methods for improving vectors and other nucleic acids used in gene therapy by
recursive
2 5 sequence recombination.
SUMMARY OF THE INVENTION
The invention provides methods of evolving nucleic acids for use in gene
therapy by recursive sequence recombination. The methods entail recombining at
least first
3 0 and second forms of the segment differing from each other in at least two
nucleotides, to
produce a library of recombinant segments. At least one recombinant segment
from the
library is then screened for a property useful in gene therapy. At least one
recombinant
2

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
segment identified by the screening is then recombined with a further form of
the segment,
the same or different from the first and second forms, to produce a further
library of
recombinant segments. The further library is then screened to identify at
least one further
recombinant segment from the further library for improvement in the property
useful for gene
therapy. Further cycles of recombination and screening are performed as
necessary until the
further recombinant segment confers a desired level of the property useful for
gene therapy.
In one embodiment, the invention provides for a method of modifying a
nucleic acid segment for use in gene therapy by recursive sequence
recombination,
comprising the following steps: (1) recombining at least a first and a second
form of the
segment differing in at least two positions, to produce a first set of
recombinant segments;
(2) screening at least one recombinant segment for a property useful in gene
therapy;
(3) recombining at least one recombinant segment generated by steps ( 1 ) and
(2) with a
variant form of the segment, the same as or different from the first or second
forms, to
produce a second set of recombinant segments; and, (4) screening at least one
recombinant
segment from the second recombination set for the property useful for gene
therapy. In a
further embodiment of this method, steps ( 1 ) to (4) are repeated until the
recursively
recombined segment confers the property useful for gene therapy. In additional
embodiments
of this method the nucleic acid segment can be a viral nucleic acid segment,
the viral nucleic
acid segment can comprise a viral vector, or at least one recombining step
occurs in vivo or in
2 0 vitro.
In one embodiment, the desired property to be acquired is improved viral
titer.
Here, the recombinant segments are screened as components of vinises by
propagation of the
viruses on cells for multiple generations and isolation of progeny viruses,
the progeny viruses
being enriched for viruses having recombinant segments conferring the property
of improved
2 5 titer.
In a second embodiment, the desired property is improved viral infectivity.
Recombinant segments can be screened as components of viruses by determining
the
percentage of a population of cells infected by a virus.
In a third embodiment, the desired property is improved expression of a gene
3 0 within the nucleic acid segment. The recombinant segments can be screened
by detecting
expression of the recombinant segments within cells.
3

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
In a fourth embodiment, the desired property is improved or altered drug
resistance. The recombinant segments can be screened by exposing the cells to
the drug and
selecting surviving cells, the surviving cells being enriched for recombinant
segments having
the property of improved or altered drug resistance.
In a fifth embodiment, the desired property is improved or altered tissue
specificity. The recombinant segments can be screened as components of viruses
by
contacting the viruses with a first population of cells for which the property
of infectivity by
the virus is desired and a second population of cells for which the property
of infectivity by
the virus is not desired, and isolating progeny virus from the first
population of cells, the
progeny viruses being enriched for recombinant segments conferring the
property of
infectivity for the first subpopulation of cells.
In a sixth embodiment, the desired property is improved packaging capacity of
a viral capsid. The recombinant segments can be screened as components of
viruses by
propagating the viruses on cells and isolating progeny viruses containing the
recombinant
segments. The packaging capacity of the viral capsid containing the
recombinant segments is
increased between successive screening steps.
In a seventh embodiment, the desired property is episomal retention. The cells
containing the recombinant segments can be screened by propagating the cells
without
selection for the recombinant segments and then propagating the cells with
selection for the
2 0 recombinant segments, the cells surviving selection being enriched for
cells harboring
recombinant segments with the property of improved episomal retention.
In an eighth embodiment, the desired property is reduced immunogenicity of
the recombinant segments or an expression product thereof. The recombinant
segments can
be screened by introducing the recombinant segments into a mammal and
recovering
2 5 surviving recombinant segments after a period of time.
In a ninth embodiment, the desired property is site-specific integration. The
recombinant segments can be screened by introducing them into cells and
recovering a region
of cellular DNA including the desired site of integration, the region being
enriched for
recombinant segments with the property of site-specific integration.
3 0 In a tenth embodiment, the desired property is increased stability. The
recombinant segments can be screened as components of viruses by subjecting
the viruses to
4

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
destabilizing conditions and recovering surviving viruses, these viruses being
enriched for
recombinant segments conferring the property.
In an eleventh embodiment, the property is capacity to confer cellular
resistance to microorganism infection. Cells containing recombinant segments
can be
screened for capacity to survive infection by the microorganism.
In a twelfth embodiment, the methods evolve vectors for introduction into
target cells in nonviral form. Recombinant segments can be selected by
introducing the
recombinant segments into a mammal, recovering cells from the mammal into
which the
segments are integrated and are expressed to produce the protein or antisense
RNA, and
recovering the recombinant segments from the cells.
In a thirteenth embodiment, the invention provides methods of improving
adenoassociated viral proteins rep and cap for expression in a packaging cell
line. Cells
containing recombinant segments of these genes are infected with a recombinant
AAV
(rAAV) containing a marker gene flanked by terminal repeat sequences (ITRs)
and a helper
virus, such as an adenovirus. The yield of progeny rAAV and helper virus
produced by
different cells are determined and cells having a high relative yield of rAAV
to helper virus
are selected.
In a fourteenth embodiment, the nucleic acid segment comprises a coding
sequence encoding a protein or antisense RNA, which can be expressed after
integration of
2 0 the segment into genomic DNA of mammalian cells.
In a fifteenth embodiment, the nucleic acid segment encodes a viral protein
and the property is capacity of a cell line containing the nucleic acid
segment to package viral
DNA transfected into the cell Line.
In a sixteenth embodiment, the nucleic acid segment encodes a DNA binding
2 5 protein, the property that is enhanced is uptake by a recipient cell of a
vector encoding the
DNA binding protein.
In a seventeenth embodiment, the invention provides an isolated recombinant
O6-methylguanine-DNA methyltransferase (MGMT) enzyme, as illustrated in Figure
5, with
the amino acid sequence of SEQ ID N0:2, encoded by the nucleic sequence of SEQ
ID NO:1.
3 0 The enzyme can have at least one amino acid segment present in a natural
human MGMT
coding sequence and absent in a natural nonhuman MGMT coding sequence, and has
at least
5

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
one amino acid segment present in the natural nonhuman MGMT coding sequence
and absent
in the natural human MGMT coding sequence. The enzyme can be a natural
nonhuman
MGMT coding sequence from mouse, rat, rabbit or hamster. The enzyme can be an
isolated
O6-methylguanine-DNA methyltransferase (MGMT) enzyme comprising a protein
encoded
by SEQ ID NO:1. In alternative embodiments, the invention provides an
expression vector
comprising the O6-methylguanine-DNA methyltransferase (MGMT) enzyme as shown
in
Figure 5 (SEQ ID NO: l ), a host cell comprising this expression vector, and a
transgenic
animal comprising this expression vector.
In another embodiment, the invention provides a method of evolving a drug
transporter gene, comprising: ( 1 ) recombinating at least first and second
forms of the gene
differing from each other in at least two nucleotides, to produce a library of
recombinant
genes;(2) screening at least one recombinant gene from the library for
conferring improved or
altered drug resistance; (3) recombining, as necessary, at least one
recombinant gene with a
further form of the gene, the same or different from the first and second
forms, to produce a
1 S further library of recombinant genes; (4} screening, as appropriate, at
least one further
recombinant gene from the further library for improved or altered drug
resistance; (5)
repeating (3) and (4), as necessary, until the further recombinant gene
confers a desired level
of improved or altered drug resistance. In this method, more than one round of
screening can
be performed between successive steps of recombining. The recombinant or
further
2 0 recombinant genes are screened by exposing cells to a drug and selecting
surviving cells, the
surviving cells being enriched for recombinant or further recombinant genes
having the
property of conferring improved or altered drug resistance. These methods also
can include
increasing the concentration of the drug between successive rounds of
screening. The drug
can be a chemotherapeutic drug. In these methods, the recombinant or further
recombinant
2 5 genes can be screened by detecting efflux from cells of a substrate for a
drug transporter
encoded by the drug transporter gene or by the recombinant or further
recombinant genes and
selecting the cells containing low intracellular amounts of said substrate.
The recombinant or
further recombinant genes can be screened by detecting influx into cells of a
substrate for a
drug transporter encoded by the drug transporter gene or by the recombinant or
further
3 o recombinant genes and selecting the cells containing high intracellular
amounts of said
substrate. In the methods, the cells can be stem cells, kidney cells, heart
cells, lung cells, liver
6

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
cells, gastrointestinal or central nervous system cells. These methods can be
for use of the
recombinant or further recombinant gene in gene therapy. The method can have
at least one
recombining step occurnng in vivo. The method can have at least one
recombining step in
vitro.
In another embodiment, the invention provides for a phagemid-adenovirus
capable of generating single stranded DNA greater than 10 kilobases comprising
an
adenovirus and a phage fl replication origin.
BRIEF DESCRIPTION OF THE FIGU F
Figure 1: Scheme for in vitro shuffling, "recursive sequence recombination,"
of genes.
Figure 2: Scheme for selecting DNA binding proteins conferring enhanced
DNA uptake by recipient cells.
Figure 3: Oligonucleotides used to generate recombinant forms of MGMT
using the recursive recombination methods of the invention.
Figure 4: Illustrates the natural diversity of five known mammalian
alkyltransferases - human, rat, mouse, hamster, and rabbit. This diversity was
used to
generate sequence diversity in the improved human MGMT gene.
Figure 5: Illustrates the nucleotide sequence (SEQ ID NO:1 ) and the amino
2 0 acid sequence (SEQ ID N0:2) of the improved human MGMT gene generated by
the
methods of the invention.
Figure 6: Illustrates the construction of an novel adenovirus-phagmid.
DEFINITIONS
2 5 The term "screening" describes what is, in general, a two-step process in
which one first determines which cells do and do not express a screening
marker and then
physically separates the cells having the desired property. Selection is a
form of screening in
which identification and physical separation are achieved simultaneously by
expression of a
selection marker, which, in some genetic circumstances, allows cells
expressing the marker to
3 0 survive while other cells die (or vice versa). Screening markers include
luciferase, beta-
galactosidase, and green fluorescent protein. Selection markers include drug
and toxin
7

CA 02268265 1999-03-24
WO 98/13485 PCT/US97117302
resistance genes. Although spontaneous selection can and does occur in the
course of natural
evolution, in the present methods selection is performed by man.
The term "exogenous DNA segment" refers to a DNA segment which is
foreign or heterologous to the cell, or homologous to the cell but in a
position within the host
cell nucleic acid in which the element is not ordinarily found. Exogenous DNA
segments are
expressed to yield exogenous polypeptides.
The term "gene" is used broadly to refer to any segment of DNA associated
with a biological function. Thus, genes include coding sequences and/or the
regulatory
sequences required for their expression. Genes also include nonexpressed DNA
segments
that, for example, form recognition sequences for other proteins.
The terms "percentage sequence identity," "sequence identity," "sequence
similarity" or "structural similarity" are calculated or determined by
comparing two optimally
aligned sequences over the window of comparison, determining the number of
positions at
which the identical nucleic acid base occurs in both sequences to yield the
number of matched
positions, dividing the number of matched positions by the total number of
positions in the
window of comparison. Optimal alignment of sequences for aligning a comparison
window
can be conducted by computerized implementations of algorithms GAP, BESTFIT,
FASTA,
and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics
Computer
Group, 575 Science Dr., Madison, WI.
2 0 The term "naturally-occurring" is used to describe an object that can be
found
in nature as distinct from being artificially produced by man. For example, a
polypeptide or
polynucleotide sequence that is present in an organism (including viruses)
that can be isolated
from a source in nature and which has not been intentionally modified by man
in the
laboratory is naturally-occurring. Generally, the term naturally-occurring
refers to an object
2 5 as present in a non-pathological (undiseased) individual, such as is
typical for the species.
The terms "isolated," "purified," or "biologically pure" refer to material
which
is substantially or essentially free from components which normally accompany
it as found in
its native state.
A nucleic acid is operably linked when it is placed into a functional
3 0 relationship with another nucleic acid sequence. For instance, a promoter
or enhancer is
operably linked to a coding sequence if it increases the transcription of the
coding sequence.
Operably linked means that the DNA sequences being linked are typically
contiguous and,
8

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
where necessary to join two protein coding regions, contiguous and in reading
frame.
However, since enhancers generally function when separated from the promoter
by several
kilobases and intronic sequences may be of variable lengths, some
polynucleotide elements
may be operably linked but not contiguous.
A specific binding affinity between two molecules, for example, a ligand and a
receptor, means a preferential binding of one molecule for another in a
mixture of molecules.
The binding of the molecules can be considered specific if the binding aff
nity is about 1 x
104 M -' to about 1 x 106 M -' or greater.
Improved drug resistance is understood to mean resistance to a higher
concentration of
the drug, irrespective of the underlying process (such as higher affinity for
the drug or
increased pump activity).
Altered drug resistance is understood to mean any alteration in the drug
resistance
profile of a cell. This includes improved drug resistance, a change in the
spectrum of drugs to
which the cell shows resistance, and decreased drug resistance.
A stem cell is understood to mean a cell of the hematopoietic system that has
the
following characteristics: ( 1 ) it has the inherent ability to differentiate
into any type of cell of
the blood cell system, and (2) it has the capacity to multiply itself without
loosing any of its
inherent characteristics.
DETAILED DESCRIPTION
2 0 I. General
The invention provides methods of evolving, i. e., modifying, a nucleic acid
for
the acquisition of or an improvement in a property or characteristic useful in
gene therapy.
The substrates for this modification, or evolution, vary in different
applications, as does the
property sought to be acquired or improved. Examples of candidate substrates
for acquisition
2 5 of a property or improvement in a property include viral and non nonviral
vectors used in
gene therapy. The methods require at least two variant forms of a starting
substrate. The
variant foams of candidate substrates can show substantial sequence or
secondary structural
similarity with each other, but they should also differ in at least two
positions. The initial
diversity between forms can be the result of natural variation, e.g., the
different variant forms
3 0 (homologs) are obtained from different individuals or strains of an
organism (including
geographic variants) or constitute related sequences from the same organism
(e.g., allelic
variations). Alternatively, the initial diversity can be induced, e.g., the
second variant form
9

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
can be generated by error-prone transcription, such as an error-prone PCR or
use of a
polymerase which lacks proof reading activity (see Liao ( 1990) Gene 88:107-11
I ), of the first
variant form, or, by replication of the first form in a mutator strain
(mutator host cells are
discussed in further detail below). The initial diversity between substrates
is greatly
augmented in subsequent steps of recursive sequence recombination.
The properties or characteristics that can be sought to be acquired or
improved
vary widely, and, of course depend on the choice of substrate. For example,
for viral and
nonviral vector sequences, improvement goals include higher titer, more stable
expression,
improved stability, higher specificity targeting, higher frequency
integration, reduced
immunogenicity of the vector sequence or an expression product thereof, and
higher
expression of gene products. For genomic DNA from a packaging cell line used
to package a
viral vector used in gene therapy, the goals of improvement include increasing
the titer of
viruses produced by the cell line.
Improvement in a property or acquisition of a property is achieved by
recursive
sequence recombination. Recursive sequence recombination can be achieved in
many
different formats and permutations of formats, as described in further detail
below. These
formats share some common principles. Recursive sequence recombination entails
successive cycles of recombination to generate molecular diversity. That is,
create a family of
nucleic acid molecules showing some sequence identity to each other but
differing in the
2 o presence of mutations. In any given cycle, recombination can occur in vivo
or in vitro,
intracellular or extracellular. Furthermore, diversity resulting from
recombination can be
augmented in any cycle by applying prior methods of mutagenesis (e.g., error-
prone PCR or
cassette mutagenesis) to either the substrates or products for recombination.
In some
instances, a new or improved property or characteristic can be achieved after
only a single
2 5 cycle of in vivo or in vitro recombination, as when using different,
variant forms of the
sequence, as homologs from different individuals or strains of an organism, or
related
sequences from the same organism, as allelic variations..
A recombination cycle is usually followed by at least one cycle of screening
or
selection for molecules having a desired property or characteristic. If a
recombination cycle
3 0 is performed in vitro, the products of recombination, i. e., recombinant
segments, are
sometimes introduced into cells before the screening step. Recombinant
segments can also be

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
linked to an appropriate vector or other regulatory sequences before
screening. Alternatively,
products of recombination generated in vitro are sometimes packaged as viruses
before
screening. If recombination is performed in vivo, recombination products can
sometimes be
screened in the cells in which recombination occurred. In other applications,
recombinant
segments are extracted from the cells, and optionally packaged as viruses,
before screening.
The nature of screening or selection depends on what property or
characteristic
is to be acquired or the property or characteristic for which improvement is
sought, and many
examples are discussed below. It is not usually necessary or desirable to
understand the
molecular basis by which particular products of recombination (recombinant
segments) have
acquired new or improved properties or characteristics relative to the
starting substrates. For
example, a gene therapy vector can have many component sequences each having a
different
intended role (e.g., coding sequence, regulatory sequences, targeting
sequences, stability-
conferring sequences, and integration sequences). Each of these component
sequences can be
varied and recombined simultaneously. Screening/selection can then be
performed, for
example, for recombinant segments that have increased stable expression in a
target cell
without the need to attribute such improvement to any of the individual
component sequences
of the vector.
Initial rounds) of screening are often performed in bacterial cells due to
high
transfection efficiencies and ease of culture. Later rounds can be performed
in mammalian
2 0 cells to optimize recombinant segments for use in an environment close to
that of their
intended use. Final rounds of screening can be performed in the precise cell
type of intended
use (e.g., a stem cell). In some instances, this stem cell can be obtained
from the patient to be
treated with a view, for example, to minimizing problems of immunogenicity in
this patient.
In some methods, use of a gene therapy vector in treatment can itself be used
as a round of
2 5 screening. That is, gene therapy vectors that are successively taken up,
integrated and/or
expressed by the intended target cells in one patient are recovered from those
target cells and
used to treat another patient. The gene therapy vectors that are recovered
from the intended
target cells in one patient are enriched for vectors that have evolved, i. e.,
have been modified
by recursive recombination, toward improved or new properties or
characteristics for specific
3 0 uptake, integration and/or expression.
The screening or selection step identifies a subpopulation of recombinant
segments that have evolved toward acquisition of a new or improved desired
property or
11

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
properties useful in gene therapy. Depending on the screen, the recombinant
segments can be
identified as components of cells, components of viruses or in free form. More
than one
round of screening or selection can be performed after each round of
recombination.
At least one and usually a collection of recombinant segments surviving
screening/selection are subject to a further round of recombination. These
recombinant
segments can be recombined with each other or with exogenous segments
representing the
original substrates or further variants thereof. Again, recombination can
proceed in vitro or in
vivo. If the previous screening step identifies desired recombinant segments
as components
of cells, the components can be subjected to further recombination in vivo, or
can be
subjected to further recombination in vitro, or can be isolated before
performing a round of in
vitro recombination. Conversely, if the previous screening step identifies
desired
recombinant segments in naked form or as components of viruses, these segments
can be
introduced into cells to perform a round of in vivo recombination. The second
round of
recombination, irrespective how performed, generates further recombinant
segments which
encompass additional diversity than is present in recombinant segments
resulting from
previous rounds.
The second round of recombination can be followed by a further round of
screening/selection according to the principles discussed above for the first
round. The
stringency of screening/selection can be increased between rounds. Also, the
nature of the
2 0 screen and the property being screened for can vary between rounds if
improvement in more
than one property is desired or if acquiring more than one new property is
desired. Additional
rounds of recombination and screening can then be performed until the
recombinant segments
have sufficiently evolved to acquire the desired new or improved property or
function.
II. Formats for Recursive Sequence Recombination
Exemplary formats and examples for using recursive sequence recombination,
sometimes referred to as DNA shuffling, sexual PCR or molecular breeding, have
been
described by the present inventors and co-workers in copending application
United States
Serial No. (USSN) 08/621,859, attorney docket no. 16528A-014612, filed March
25, 1996;
3 0 international application PCT/LJS95/02126, filed February 17, 1995,
published as WO
95/22625; Stemmer (1995) Science 270:1510; Stemmer (1995) Gene 164:49-53;
Stemmer
(1995) BiolTechnolo~ 13:549-553; Stemmer (1994) Proc. Natl. Acad. Sci. USA
91:10747-
12

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
10751; Stemmer (1994) Nature 370:389-391; Crameri (1996) Nature Medicine 2:1-
3;
Crameri ( 1996) Nature Biotechnology 14:3 I S-3 I 9.
( ~~ In Vitro Formats
One embodiment for shuffling DNA sequences in vitro is illustrated in Fig. 1.
The initial substrates for recombination are a pool of related sequences,
e.g., different, variant
forms, as homologs from different individuals or strains of an organism, or
related sequences
from the same organism, as allelic variations. The X's in the Fig. 1, panel A,
show where the
sequences diverge. The sequences can be DNA or RNA and can be of various
lengths
depending on the size of the gene or DNA fragment to be recombined or
reassembled.
1 o Preferably the sequences are from 50 base pairs (bp) to 50 kilobases (kb).
The pool of related substrates are converted into overlapping fragments, e.g.,
from about 5 by to 5 kb or more, as shown in Fig. 1, panel B. Often, for
example, the size of
the fragments is from about 10 by to 1000 bp, and sometimes the size of the
DNA fragments
is from about 100 by to 500 bp. The conversion can be effected by a number of
different
methods, such as DNase I or RNAse digestion, random shearing or partial
restriction enzyme
digestion. For discussions of protocols for the isolation, manipulation,
enzymatic digestion,
and the like of nucleic acids, see, for example, Sambrook, MOLECULAR CLONING:
A
LABORATORY MANUAL (2ND ED.), Vols. I-3, Cold Spring Harbor Laboratory, (1989)
(Sambrook), and, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. Greene
Publishing and Wiley-Interscience, New York (1987) (Ausubel). The
concentration of
nucleic acid fragments of a particular length and sequence is often less than
0.1 % or 1 % by
weight of the total nucleic acid. The number of different specific nucleic
acid fragments in
the mixture is usually at least about 100, 500 or 1000.
The mixed population of nucleic acid fragments are converted to at least
partially single-stranded form using a variety of techniques, including, for
example, heating,
chemical denaturation, use of DNA binding proteins, and the like. Conversion
can be
effected by heating to about 80°C to 100°C, more preferably from
90°C to 96°C, to form
single-stranded nucleic acid fragments and then reannealing. Conversion can
also be effected
by treatment with single-stranded DNA binding protein (see Wold ( 1997) Annu.
Rev.
Biochem. 66:61-92) or recA protein (see Kiianitsa (1997) Proc. Natl. Acad.
Sci. USA
94:7837-7840). Single-stranded nucleic acid fragments having regions of
sequence identity
13

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
with other single-stranded nucleic acid fragments can then be reannealed by
cooling to 20°C
to 75°C, and preferably from 40°C to 65°C. Renaturation
can be accelerated by the addition
of polyethylene glycol (PEG), other volume-excluding reagents or salt. The
salt
concentration is preferably from 0 mM to 200 mM, more preferably the salt
concentration is
from 10 mM to 100 mM. The salt may be KCl or NaCI. The concentration of PEG is
preferably from 0% to 20%, more preferably from 5% to 10%. The fragments that
reanneal
can be from different substrates as shown in Fig. 1, panel C. The annealed
nucleic acid
fragments are incubated in the presence of a nucleic acid polymerise, such as
Taq or Klenow,
and dNTP's (i.e. dATP, dCTP, dGTP and dTTP). If regions of sequence identity
are large,
Taq polymerise can be used with an annealing temperature of between 45-
65°C. If the areas
of identity are small, Klenow polymerise can be used with an annealing
temperature of
between 20-30°C. The polymerise can be added to the random nucleic acid
fragments prior
to annealing, simultaneously with annealing or after annealing.
The process of denaturation, renaturation and incubation in the presence of
polymerise of overlapping fragments to generate a collection of
polynucleotides containing
different permutations of fragments is sometimes referred to as shuffling of
the nucleic acid
in vitro. This cycle is repeated for a desired number of times. Preferably the
cycle is repeated
from 2 to 100 times, more preferably the sequence is repeated from 10 to 40
times. The
resulting nucleic acids are a family of double-stranded polynucleotides of
from about 50 by to
about 100 kb, preferably from 500 by to 50 kb, as shown in Fig. l, panel D.
The population
represents variants of the starting substrates showing substantial sequence
identity thereto but
also diverging at several positions. The population has many more members than
the starting
substrates. The population of fragments resulting from shuffling is used to
transform host
cells, optionally after cloning into a vector.
2 5 In one embodiment utilizing in vitro shuffling, subsequences of
recombination substrates can be generated by amplifying the full-length
sequences under
conditions which produce a substantial fraction, typically at least 20 percent
or more, of
incompletely extended amplification products. Another embodiment uses random
primers to
prime the entire template DNA to generate less than full length amplification
products. The
3 0 amplification products; including the incompletely extended amplification
products are
denatured and subjected to at least one additional cycle of reannealing and
amplification.
This variation, in which at least one cycle of reannealing and amplification
provides a
14

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
substantial fraction of incompletely extended products, is termed
"stuttering." In the
subsequent amplification round, the partially extended (less than full length)
products
reanneal to and prime extension on different sequence-related template
species. In another
embodiment, the conversion of substrates to fragments can be effected by
partial PCR
amplification of substrates.
In another embodiment, a mixture of fragments is spiked with one or more
oiigonucleotides. The oligonucleotides can be designed to include
precharacterized mutations
of a wildtype sequence, or sites of natural variations between individuals or
species. The
oligonucleotides also include sufficient sequence or structural homology
flanking such
1 o mutations or variations to allow annealing with the wildtype fragments.
Annealing
temperatures can be adjusted depending on the length of homology.
In a further embodiment, recombination occurs in at least one cycle by
template switching, such as when a DNA fragment derived from one template
primes on the
homologous position of a related but different template. Template switching
can be induced
by addition of recA (see Kiianitsa ( I 997) supra), rad5 l (see Namsaraev (
1997) Mol. Cell.
Biol. 17:5359-5368), rad55 (see Clever ( 1997) EMBO J. 16:2535-2544), rad57
(see Sung
(1997) Genes Dev. 11:1111-1121 ) or other polymerises (e.g., viral
polymerises, reverse
transcriptase) to the amplification mixture. Template switching can also be
increased by
increasing the DNA template concentration.
2 o Another embodiment utilizes at least one cycle of amplification, which can
be
conducted using a collection of overlapping single-stranded DNA fragments of
related
sequence, and different lengths. Fragments can be prepared using a single
stranded DNA
phage, such as M13 (see Wang (1997) Biochemistry 36:9486-9492). Each fragment
can
hybridize to and prime polynucleotide chain extension of a second fragment
from the
2 5 collection, thus forming sequence-recombined polynucleotides. In a further
variation, ssDNA
fragments of variable length can be generated from a single primer by Pfu,
Taq, Vent, Deep
Vent, UlTma DNA polymerise or other DNA polymerises on a first DNA template
(see
Cline (1996) Nucleic Acids Res. 24:3546-3551). The single stranded DNA
fragments are
used as primers for a second, Kunkel-type template, consisting of a uracil-
containing circular
3 0 ssDNA. This results in multiple substitutions of the first template into
the second. See
Levichkin (1995) Mol. Biology 29:572-577; Jung (1992) Gene 121:17-24.

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
Reintroduction of Genes Shuffled in vitro into Cells
In a further embodiment, whole cells and organisms can be improved by
evolving a transgene within those cells and organisms by recursive cycles of
in vitro
shuffling. The transgene is subjected to the recursive recombination methods
of the
invention, and the shuffled sequence library is put back into the
cell/organism for selection.
While this method is useful if multiple copies of the modified transgene are
reintegrated into
a cell, in a preferred variation of this selection assay, only a single copy
of the modified
transgene is inserted into each cell. Another preferred variation of this
selection assay
involves reducing the transcriptional expression variability of the modified
transgene that
1 o may result from differences in chromosomal location of integration sites.
This requires a
means for defined, site-specific integration of the modified transgene. These
methods can
also be used to evolve an episomal vector (which can replicate inside the
celi) which can site-
specifically integrate into a chromosome.
Use of retroviruses to shuttle the modified transgene back into the cell for
selection has the advantage that they integrate as a single copy. However,
this insertion is not
site-specific, i.e., the retrovirus inserts in a random location in the
chromosome.
Adenoviruses and ars-plasmids are also used to shuttle modified transgenes,
however, they
integrate as multiple copies. While wild type AAV integrates as a single copy
in
chromosome q 19, commonly used modified versions of AAV do not. Homologous
2 0 recombination is also used to insert a modified recombinant segment
(transgene) into a
chromosome, but this method can be inefficient and may result in the
integration of two
copies in the pair of chromosomes. To solve these problems, one embodiment of
the
invention utilizes site-specific integration systems to target the transgene
to a specific,
constant location in the genome. A preferred embodiment uses the Cre/LoxP or
the related
2 5 FLP/FRT site-specific integration system. The Cre/LoxP system uses a Cre
recombinase
enzyme to mediate site-specific insertion and excision of viral or phage
vectors into a specific
palindromic 34 base pair sequence called a "LoxP site." Lox P sites can be
inserted to a
mammalian genome of choice, to create, for example, a transgenic animal
containing the Lox
P site, by homologous recombination (see Rohlmann (1996) Nature Biotech.
14:1562-1565).
3 0 If a genome is engineered to contain a LoxP site in a desired location,
infection of such cells
with vectors carrying a gene for the Cre recombinase results in the efficient,
site-specific
16

CA 02268265 1999-03-24
WO 98113485 PCT/ITS97/17302
integration of the transgene-containing vector into the LoxP site. This
approach is
reproducible from cycle to cycle and provides a single copy of the modified
transgene
(recombinant sequence) at a constant, defined location. Thus, a transgene of
interest can be
modified using the recursive sequence recombination methods of the invention
in vitro and
reinserted into the cell for in vivolin situ selection for the new or improved
property in the
optimal way with minimal noise. This technique can also be used in vivo, as
discussed
below. See, example, Agah (1997) J. Clin. Invest. 100:169-179; Akagi (1997)
Nucleic Acids
Res. 25:1766-1773; Xiao (1997) Nucleic Acids Res 25:2985-2991; Jiang (1997)
Curr Biol
7:321-8323, Rohlmann (1996) Nature Biotech. 14:1562-1565; Siegal (1996)
Genetics 144:
l0 715-726; Wild (1996) Gene 179:181-I 88. The evolution of Cre is discussed
in further detail,
below.
f2) In Trivo Formats
(a) Plasmid-Plasmid Recombination
The recursive recombination methods of the invention include plasmid-
plasmid recombinations. In this and other embodiments, the initial substrates
for
recombination are a collection of polynucleotides comprising variant forms of
nucleic acid of
interest, such as a gene, a vector, a transcriptional regulatory sequence, or
the like. The
variant forms can have substantial sequence identity to each other; for
example, sequence
identity sufficient to allow homologous recombination between substrates (see
Datta ( 1997)
2 0 Proc. Natl. Acad. Sci. USA 94:9757-9762; Shimizu ( 1997) J. Mol. Biol.
266:297-305; Watt
(1985) Proc. Natl. Acad. Sci. USA 82:4768-4772). The diversity between the
polynucleotides
can be naturai (e.g., allelic or species variants), induced (e. g., irr vitro
generated, as by error-
prone PCR, see Light (1995) Bioorg. Med. Chem. 3:955-967), or the result of in
vitro
recombination. Diversity can also result from resynthesizing genes encoding
natural proteins
2 5 with alternative and/or mixed codon usage. There should be at least
sufficient diversity
between substrates that recombination can generate more diverse products than
there are
starting materials. There must be at least two substrates differing in at
least two positions.
However, in another embodiment, a library of substrates of 103-108 members is
employed.
The degree of diversity depends on the length of the substrate being
recombined and the
3 o extent of the functional change to be evolved. Diversity at between 0.1-
50% of positions is
typical.
17

CA 02268265 1999-03-24
WO 98/13485 PCT/US97117302
The diverse initial substrates or recombinant segments modified by the
methods of the invention can be incorporated into plasmids. In one embodiment,
the
plasmids are standard cloning vectors, e.g., bacterial multicopy plasmids.
However, in
alternative embodiments, described below, the plasmids include mobilization
functions. The
initial substrates or recombinant segments can be incorporated into the same
or different
plasmids. Often at least two different types of plasmid having different types
of selection
marker are used to allow selection for cells containing at least two types of
vector. Also,
where different types of plasmid are employed, the different plasmids can come
from two
distinct incompatibility groups to allow stable co-existence of two different
plasmids within
the cell. Nevertheless, plasmids from the same incompatibility group can still
co-exist within
the same cell for sufficient time to allow homologous recombination to occur.
Plasmids containing diverse substrates are initially introduced into
procaryotic
or eukaryotic cells by any transfection methods, e.g., chemical
transformation, natural
competence, electroporation, viral transduction or biolistics (see, for
example, Sambrook for
a detailed descriptions of introducing DNA into cells; Hapala (1997) Crit.
Rev. Biotechnol.
17:105-122). Often, the plasmids are present at or near saturating
concentration (with respect
to maximum transfection capacity) to increase the probability of more than one
plasmid
entering the same cell. The plasmids containing the various substrates or
recombinant
segments can be transfected simultaneously or in multiple rounds. For example,
in the latter
2 0 approach cells can be transfected with a first aliquot of plasmid,
transfectants selected and
propagated, and then infected with a second aliquot of plasmid.
Having introduced the plasmids into cells, recombination between substrates
to generate recombinant genes or other nucleic acid segments occurs within
cells containing
multiple different plasmids merely by propagating the plasmids in the cells.
However, cells
2 5 that receive only one plasmid are unable to participate in recombination
and the potential
contribution of substrates on such plasmids to evolution (sequence
modification) is not fully
exploited, although these plasmids may contribute to new sequence diversity if
they are
propagated in mutator cells (described below) or otherwise accumulate point
mutations (i. e.,
by ultraviolet radiation treatment). The rate of evolution, i. e., modif
cation of nucleic acid
3 0 sequence by the methods of the invention, can be increased by allowing all
substrates to
participate in recombination. In one embodiment, this is achieved by
subjecting transfected
18

CA 02268265 1999-03-24
WO 98/13485 PC'T/US97/I7302
cells to electroporation. The conditions for electroparation are the same as
those
conventionally used for introducing exogenous DNA into cells (e.g., 1,000-
2,500 volts, 400
~F and a 1-2 mM gap). Under these conditions, plasmids are exchanged between
cells
allowing all substrates to participate in recombination. In addition the
products of
recombination can undergo further rounds of recombination with each other or
with the
original substrate.
In another embodiment, the rate of evolution, i.e., the rate of recursive
sequence modification, can also be increased by use of conjugative transfer.
To exploit
conjugative transfer, substrates are cloned into plasmids having MOB genes,
and tra genes
are also provided in cis or in trans to the MOB genes. The effect of
conjugative transfer is
very similar to electroporation in that it allows plasmids to move between
cells and allows
recombination between any substrate, and the products of previous
recombination to occur
merely by propagating the culture. The details of how conjugative transfer is
exploited in
these vectors are discussed in more detail below (see also Cabezon (1997) Mol.
Gen. Genet.
254:400-406.)
The rate of evolution can also be increased by fusing cells to induce exchange
of plasmids or chromosomes. Fusion can be induced by chemical agents, such as
PEG, or
viruses or viral proteins, such as influenza virus hemagglutinin, HSV-1 gB and
gD, or
fusigenic liposomes (see Dzau (1996) Proc. Natl. Acad. ,Sci. USA 93:11421-
11425).
2 0 The rate of evolution can also be increased by use of mutator host cells;
e.g.,
bacterial Mut L, S, D, T, H mutator cells, insect (Drosophila) and mouse
mutator cells, and
human cell lines with defective DNA repair mechanisms, such as those from
Ataxia
telangiectasia patients, see Morgan (1997) Cancer Res. 57:3386-3389; Greener
(1997) Mol.
Biotechnol. 7:189-195; Mason (1997) Genetics 146:1381-1397; Aronshtam (1996)
Nucleic
Acids Res 24:2498-2504; Seong (1995) Int. J. Radiat. Oncol. Biol. Phys.33:869-
874; Wu
(1994) J. Bacteriol. 176:5393-5400; Rewinski (1987) Nucleic Acids Res.15:8205-
8215;
Aizawa (1986) Jpn. J. Cancer Res. 77:327-329.
The time for which cells are propagated and recombination is allowed to
occur, of course, varies with the cell type but is generally not critical,
because even a small
3 0 degree of recombination can substantially increase diversity relative to
the starting materials.
Cells bearing plasmids containing recombined genes are subject to screening or
selection for
a desired function. For example, if the substrate being evolved contains a
drug resistance
19

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
gene, one selects for drug resistance. In the case of drug resistance genes
which encode drug
transporteres flow cytometry can be employed to enrich for cells exhibiting
high levels of a
mutant transporter phenotype by screening for drug efflux. This is done by
employing
fluorescent transporter substrates or fluorescent analogues of the drug
substrate in question.
Specifically substrates that are poor substrates for the wildtype transporter
are used. Sorting
those cells exhibiting low levels of fluorescence will result in enrichment of
cells expressing a
mutant gene encoding a transporter pumping the substrate used. Cells surviving
screening or
selection or cells enriched by flow cytometry can be subjected to one or more
rounds of
screening/selection followed by recombination or can be subjected directly to
an additional
round of recombination.
The next round of recombination can be achieved by several different formats
independently of the previous round. For example, in one embodiment, a further
round of
recombination can be effected simply by resuming (repeating) the
electroporation or
conjugation-mediated intercellular transfer of plasmids described above. In
another
embodiment, a fresh substrate or substrates, the same or different from
previous substrates,
can be transfected into cells surviving selection/screening. The new
substrates can be
included in plasmid vectors bearing a different selective (selection) markers)
and/or from a
different incompatibility group than the original plasmids. Selection markers
confer a
selectable phenotype on transformed cells. For example, the marker may encode
antibiotic
2 0 resistance, particularly resistance to chloramphenicol, kanamycin, G4I 8,
bleomycin and
hygromycin, to permit selection of those cells transformed with the desired
DNA sequences,
see for example, Blondelet-Rouault ( 1997) Gene 190:315-317. Because
selectable marker
genes conferring resistance to substrates like neomycin or hygromycin can only
be utilized in
tissue culture, chemoresistance genes are also used as selectable markers in
vitro and in vivo.
Various target cells are rendered resistant to anticancer drugs by transfer of
chemoresistance
genes encoding P-glycoprotein, multidrug resistance-associated protein-
transporter,
dihydrofolate reductase, glutathione -S-transferase, O 6-alkylguanine DNA
alkyltransferase
(Tano (1997) J. Biol. Chem. 272:13250-13254), or aldehyde reductase (Licht
(1997) Stem
Cells 15:104-111 ) and the like.
3 0 As a further embodiment, cells surviving selection/screening can be
subdivided into two subpopulations, and plasmid DNA from one subpopulation
transfected

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
into the other, where the substrates from the plasmids from the two
subpopulations undergo a
further round of recombination. In either of the latter two embodiments, the
rate of evolution
can be increased by employing any of the techniques described above, including
DNA
extraction, electroporation, conjugation or use of mutator cells. In a still
further embodiment,
DNA from cells surviving screening/selection can be extracted and subjected to
in vitro DNA
shuffling.
After the second round of recombination, a further round of screening/
selection can be performed. 1n one embodiment, the screening or selection is
performed
under conditions of increased stringency. If desired, further rounds of
recombination and
selection/screening can be performed using the same strategies as used in the
second round.
With successive rounds of recombination and selection/screening, the surviving
recombined
substrates evolve toward acquisition of a desired phenotype or characteristic.
Typically, in
this and other recursive recombination methods of the invention, the final
product of
recombination that has acquired the desired phenotype can differ from starting
(initial)
substrates at 0.1 %-25% of positions. The methods of the invention can
evolve/modify
nucleic acid sequences at a rate orders of magnitude in excess (e.g., by at
least 10-fold, 100-
fold, 1000-fold, or 10,000 fold) of the rate calculated for naturally acquired
mutation (about 1
mutation per 10-9 positions per generation, see Anderson ( 1996) Proc. Natl.
Acad. Sci. USA
93:906-907).
2 0 (bl Virus-Piasmid Recombination
The recursive recombination methods of the invention include virus-plasmid
recombinations. The strategy used for plasmid-plasmid recombination can also
be used for
other embodiments of the invention, including virus-plasmid recombination or
phage-plasmid
recombination. However, some additional comments particular to the use of
viruses are
2 5 appropriate. The initial substrates for recombination are cloned into both
plasmid and viral
vectors. It is usually not critical which substrates) are inserted into the
viral vector and
which into the plasmid, although usually the viral vector should contain
different substrates)
from the plasmid. As before, the plasmid (and the virus) typically contains a
selective
marker. The plasmid and viral vectors can both be introduced into cells by
transfection as
3 0 described above. However, a more efficient procedure is to transfect the
cells with plasmid,
select transfectants and infect the transfectants with virus. Because the
efficiency of infection
21

CA 02268265 1999-03-24
WO 98/13485 PCT/US97117302
of many viruses approaches 100% for many cells, most cells transfected and
infected by this
route contain both a plasmid and virus bearing different substrates.
Homologous recombination occurs between plasmid and virus generating both
recombined plasmids and recombined virus. For some viruses, such as
filamentous phage, in
which intracellular DNA exists in both double-stranded and single-stranded
forms, both can
participate in recombination. Provided that the virus is not one that rapidly
kills cells,
recombination can be augmented by use of electroporation or conjugation to
transfer plasmids
between cells. Recombination can also be augmented for some types of virus by
allowing the
progeny virus from one cell to reinfect other cells. For some types of virus,
virus infected-
cells show resistance to superinfection. However, such resistance can be
overcome by
infecting at high multiplicity and/or using mutant strains of the virus in
which resistance to
superinfection is reduced.
The result of infecting plasmid-containing cells with virus depends on the
nature of the virus. Some viruses, such as filamentous phage, stably exist
with a plasmid in
the cell and also extrude progeny phage from the cell (see Russel (1997) Gene
192:23-32).
Other viruses, such as lambda having a cosmid genome, stably exist in a cell
like piasmids
without producing progeny virions. Other viruses, such as the T-phage and
lytic lambda,
undergo recombination with the plasmid but ultimately kill the host cell and
destroy plasmid
DNA. For viruses that infect cells without killing the host, cells containing
recombinant
2 0 plasmids and virus can be screened/selected using the same approach as for
piasmid-plasmid
recombination. Progeny virus extruded by cells surviving selection/screening
can also be
collected and used as substrates in subsequent rounds of recombination. For
viruses that kill
their host cells, recombinant genes resulting from recombination reside only
in the progeny
virus. If the screening or selective assay requires expression of recombinant
genes in a cell,
2 5 the recombinant genes should be transferred from the progeny virus to
another vector, e.g., a
plasmid vector, and retransfected into cells before selection/screening is
performed.
For filamentous phage, the products of recombination are present in both cells
surviving recombination and in phage extruded from these cells. The dual
source of
recombinant products provides some additional options relative to the plasmid-
plasmid
3 0 recombination. In one embodiment, DNA can be isolated from phage particles
for use in a
round of in vitro recombination. In an alternative embodiment, the progeny
phage can be
22

CA 02268265 1999-03-24
WO 98/13485 PCT/US97117302
used to transfect or infect cells surviving a previous round of
screening/selection, or fresh
cells transfected with fresh substrates for recombination.
(cl Virus-Virus Recombination
The recursive recombination methods of the invention also include virus-virus
recombinations. The principles described for plasmid-plasmid and plasmid-viral
recombination can be applied to virus-virus recombination with a few
modifications. The
initial substrates for recombination are cloned into a viral vector. In a
preferred embodiment,
the same vector is used for all substrates. Preferably, the virus is one that,
naturally or as a
result of mutation, does not kill cells. After insertion, some viral genomes
can be packaged in
vitro. The packaged viruses are used to infect cells at high multiplicity such
that there is a
high probability that a cell receives multiple viruses bearing different
substrates.
After the initial round of infection, subsequent steps depend on the nature of
infection, as discussed in the previous section. For example, if the viruses
have phagemid
genomes such as lambda cosmids or M13, F1 or Fd phagemids, the phagemids
behave as
plasmids within the cell and undergo recombination simply by propagating the
cells.
Recombination is particularly efficient between single-stranded forms of
intracellular DNA.
Recombination can be augmented by electroporation of cells. Following
selection/screening,
cosmids containing recombinant genes can be recovered from surviving cells
(e.g., by heat
induction of a cos- lysogenic host cell), repackaged in vitro, and used to
infect fresh cells at
2 0 high multiplicity for a further round of recombination.
If the viruses are filamentous phage, recombination of replicating form DNA
occurs by propagating the culture of infected cells. Selection/screening
identifies colonies of
cells containing viral vectors having recombinant genes with improved
properties, together
with phage extruded from such cells. Subsequent options are essentially the
same as for
2 5 plasmid-viral recombination.
(d) Chromosome-Plasmid Recombinat»n
The recursive recombination methods of the invention also include
chromosome-plasmid recombinations. This format can be used to evolve both the
chromosomal and plasmid-borne substrates. The format is particularly useful in
situations in
3 0 which many chromosomal genes contribute to a phenotype or one does not
know the exact
location of the chromosomal genes) to be evolved. The initial substrates for
recombination
are cloned into a plasmid vector. If the chromosomal genes) to be evolved are
known, the
23

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
substrates constitute a family of sequences showing a high degree of sequence
identity but
some divergence from the chromosomal gene. If the chromosomal genes to be
evolved have
not been located, the initial substrates usually constitute a library of DNA
segments of which
only a small number show sequence identity to the gene or genes) to be
evolved. Divergence
between plasmid-borne substrate and the chromosomal genes) can be induced by
mutagenesis or by obtaining the plasmid-borne substrates from a different
species than that of
the cells bearing the chromosome, as discussed above.
The plasmids bearing substrates for recombination are transfected into cells
having chromosomal genes) to be evolved/ modified to acquire a new or modified
property.
Evolution by recursive recombination can occur simply by propagating the
culture. In
another embodiment, the nucleic acid sequence modification can be accelerated
by
transferring plasmids between cells by conjugation or electroporation. In a
further
embodiment, evolution by recursive recombination can be further accelerated by
use of
mutator host cells or by seeding a culture of nonmutator host cells being
evolved with mutator
host cells and inducing intercellular transfer of plasmids by electroporation
or conjugation.
Preferably, mutator host cells used for seeding contain a negative selection
marker to
facilitate isolation of a pure culture of the nonmutator cells being evolved.
Selection/screening identifies cells bearing chromosomes and/or plasmids that
have evolved
toward acquisition or modification of a desired property or function.
2 0 Subsequent rounds of recombination and selection/screening proceed in
similar fashion to those described for plasmid-plasmid recombination. For
example, further
recombination can be effected by propagating cells surviving recombination in
combination
with electroporation or conjugative transfer of plasmids. Alternatively,
plasmids bearing
additional substrates for recombination can be introduced into the surviving
cells. Preferably,
2 5 such plasmids are from a different incompatibility group and bear a
different selective marker
than the original plasmids to allow selection for cells containing at least
two different
plasmids. As a further alternative, plasmid and/or chromosomal DNA can be
isolated from a
subpopulation of surviving cells and transfected into a second subpopulation.
Chromosomal
DNA can be cloned into a plasmid vector before transfection.
3 0 (e) Virus-Chromosome Recombination
The recursive recombination methods of the invention also include
chromosome-virus recombinations. As in previously described embodiments, the
virus is
24

CA 02268265 1999-03-24
WO 98/13485 PCTlUS97l17302
usually one that does not kill the cells, and is often a phage or phagemid.
The procedure is
substantially the same as for plasmid-chromosome recombination. Substrates for
recombination are cloned into the vector. Vectors including the substrates can
then be
transfected into cells or in vitro packaged and introduced into cells by
infection. Viral
genomes recombine with host chromosomes merely by propagating a culture.
Evolution can
be accelerated by allowing intercellular transfer of viral genomes by
electroporation, or
reinfection of cells by progeny virions. Screening/selection identifies cells
having
chromosomes and/or viral genomes that have evolved toward acquisition of a new
or
modified property or desired function.
There are several options for subsequent rounds of recombination. For
example, viral genomes can be transferred between cells surviving
selection/recombination
by electroporation. Alternatively, viruses extruded from cells surviving
selection/screening
can be pooled and used to superinfect the cells at high multiplicity.
Alternatively, fresh
substrates for recombination can be introduced into the cells, either on
plasmid or viral
vectors.
III. Vectors Used in Gene Therap,Y
The invention provides for methods of modifying a vector by recursive
recombination for use in gene therapy. Broadly speaking, a gene therapy vector
is an
exogenous polynucieotide which produces a medically useful phenotypic effect
upon the
mammalian cells) into which it is transferred. A vector may or may not have an
origin of
replication. For example, it is useful to include an origin of replication in
a vector for
propagation of the vector prior to administration to a patient. However, the
origin of
replication can often be removed before administration if the vector is
designed to integrate
into host chromosomal DNA or bind to host mRNA or DNA. Vectors used in gene
therapy
can be viral or nonviral and include but are not restricted to those described
for AAV vectors
in patent applications PCT/hTL96/00472 filed November 29 1996, for retrovirus
vectors in
patent application PCT/NL96/00195 filed May 7 1996 (published as W096/35798),
for
adenovirus vectors in patent application EP-A-95202213 filed August 15 1995
and for
3 0 nonvital gene transfer PCT/NL96/00324 filed August 16 1996.
Viral vectors are usually introduced into a patient as components of a virus.
Illustrative vectors incorporating nucleic acids to be modified by the
recursive recombination

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
methods of the invention include, for example, adenovirus-based vectors
(Cantwell (1996)
Blood 88:4676-4683; Ohashi (1997) Proc Natl Acad Sci USA 94:1287-1292),
Epstein-Barr
virus-based vectors (Mazda ( 1997) J Immunol Methods 204:143-1 S 1 ),
adenovirus-associated
virus vectors, Sindbis virus vectors (Strong ( 1997) Gene Ther 4: 624-627),
herpes simplex
virus vectors (Kennedy (1997) Brain 120: 1245-1259) and retroviral vectors
(Schubert (1997)
Curr Eye Res 16:656-662) .
Nonviral vectors, typically dsDNA, can be transferred as naked DNA or
associated with a transfer-enhancing vehicle, such as a receptor-recognition
protein,
liposome, lipoamine, or cationic lipid. This DNA can be transferred into a
cell using a
1 o variety of techniques well known in the art. For example, naked DNA can be
delivered by
the use of liposomes which fuse with the cellular membrane or are endocytosed,
i.e., by
employing ligands attached to the liposome, or attached directly to the DNA,
that bind to
surface membrane protein receptors of the cell resulting in endocytosis.
Alternatively, the
cells may be permeabilized to enhance transport of the DNA into the cell,
without injuring the
host cells. One can use a DNA binding protein, e.g., HBGF-1, known to
transport DNA into
a cell. These procedures for delivering naked DNA to cells are useful in vivo.
For example,
by using liposomes, particularly where the liposome surface carries iigands
specific for target
cells, or are otherwise preferentially directed to a specific organ, one may
provide for the
introduction of the DNA into the target cells/organs in vivo.
2 0 A. Viral-Based Methods
Various viral vectors, such as retroviruses, adenoviruses, adenoassociated
viruses and herpes viruses, are used in gene therapy. They are often made up
of two
components, a modified viral genome and a coat structure surrounding it (see
generally Smith
(1995} Annu. Rev. Microbiol. 49, 807-838), although sometimes viral vectors
are introduced
2 5 in naked form or coated with proteins other than viral proteins. Most
current vectors have
coat structures similar to a wildtype virus. This structure packages and
protects the viral
nucleic acid and provides the means to bind and enter target cells. However,
the viral nucleic
acid in a vector designed for gene therapy can be changed in many ways. The
goals of these
changes are to disable growth of the virus in target cells while maintaining
its ability to grow
3 0 in vector form in available packaging or helper cells, to provide space
within the viral
genome for insertion of exogenous DNA sequences, and to incorporate new
sequences that
26

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
encode and enable appropriate expression of the gene of interest. Thus, vector
nucleic acids
generally comprise two components: essential cis-acting viral sequences for
replication and
packaging in a helper line and the transcription unit for the exogenous gene.
Other viral
functions are expressed in traps in a specific packaging or helper cell line.
( 11 Retroviruses
Retroviruses comprise a large class of enveloped viruses that contain single--
stranded RNA as the viral genome. During the normal viral life cycle, viral
RNA is reverse-
transcribed to yield double-stranded DNA that integrates into the host genome
and is
expressed over extended periods. As a result, infected cells shed virus
continuously without
apparent harm to the host cell. The viral genome is small (approximately I 0
kb), and its
prototypical organization is extremely simple, comprising three genes encoding
gag, the
group specific antigens or core proteins; pol, the reverse transcriptase; and
env, the viral
envelope protein. The termini of the RNA genome are called long terminal
repeats (LTRs)
and include promoter and enhancer activities and sequences involved in
integration. The
genome also includes a sequence required for packaging viral RNA and splice
acceptor and
donor sites for generation of the separate envelope mRNA. Most retroviruses
can integrate
only into replicating cells, although human immunodeficiency virus (HIV)
appears to be an
exception. This property can restrict the use of retroviruses as vectors for
gene therapy.
Retrovirus vectors are relatively simple, containing the S' and 3' LTRs, a
2 0 packaging sequence, and a transcription unit composed of the gene or genes
of interest, which
is typically an expression cassette. Useful vectors have been described in
PCT/NL96/00195
filed May 7 1996 disclosing vectors having mutant LTRs with the wildtype
enhancer
sequences replaced by a mutant polyoma enhancer sequence.
To grow such a vector, one must provide the missing viral functions in traps
2 5 using a so-called packaging cell line. Such a cell is engineered to
contain integrated copies of
gag, pol, and env but to lack a packaging signal so that no helper virus
sequences become
encapsidated. Additional features added to or removed from the vector and
packaging cell
line reflect attempts to render the vectors more efficacious or reduce the
possibility of
contamination by helper virus.
3 0 The main advantage of retroviral vectors is that they integrate in the
chromosome and are therefore potentially capable of long-term expression. They
can be
grown in relatively large amounts, but care is needed to ensure the absence of
helper virus.
27

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
(2) Adenoviruses
Adenoviruses comprise a large class of nonenveloped viruses containing linear
double-stranded DNA. The normal life cycle of the virus does not require
dividing cells and
involves productive infection in permissive cells during which large amounts
of virus
accumulate. The productive infection cycle takes about 32-36 hours in cell
culture and
comprises two phases, the early phase, prior to viral DNA synthesis, and the
late phase,
during which structural proteins and viral DNA are synthesized and assembled
into virions.
In general, adenovirus infections are associated with mild disease in humans.
Adenovirus vectors are somewhat larger and more complex than retrovirus or
AAV vectors, partly because only a small fraction of the viral genome is
removed from most
current vectors. If additional genes are removed, they are provided in trans
to produce the
vector, which so far has proved difficult. Instead, two general types of
adenovirus-based
vectors have been studied, E3-deletion and EI-deletion vectors. Some viruses
in laboratory
stocks of wild-type lack the E3 region and can grow in the absence of helper.
This ability
does not mean that the E3 gene products are not necessary in the wild, only
that replication in
cultured cells does not require them. Deletion of the E3 region allows
insertion of exogenous
DNA sequences to yield vectors capable of productive infection and the
transient synthesis of
relatively large amounts of encoded protein.
Deletion of the E1 region disables the adenovirus, but such vectors can still
be
2 o grown because there are several human cell lines (called 293, 911 and
PER.C6) are available
that constitutively express the E 1 region of AdS. Most recent gene therapy
applications
involving adenovirus have utilised E1 replacement vectors grown in PERC6 cells
disclosed in
PCT/NL96/00244 filed June I4 1996 (published as W097/00326). PerC6 produced
recombinant adenovirus lots carrying for example the HSV thymidine kinase gene
do not
2 S contain any detectable levels of replication competent adenovirus (RCA)
and are therefore
preferred for use in gene therapy and thus are an embodiment of the present
invention.
The main advantages of adenovirus vectors are that they are capable of
efficient episomal gene transfer in a wide range of cells and tissues and that
they are easy to
grow in large amounts. The main disadvantage is that the host response to the
virus appears
3 0 to limit the duration of expression and the ability to repeat dosing, at
least with high doses of
first-generation vectors.
28

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
In another embodiment, the recursive recombination methods of the invention
are used to construct a novel adenovirus-phagmid capable of packaging DNA
inserts over 10
kilobases in size. Incorporation of a phage fl origin in a plasmid using the
methods of the
invention also generates a novel in vivo shuffling format capable of evolving
whole genomes
of viruses, such as the 36 kb family of human adenoviruses. The widely used
human
adenovirus type 5 (Ad5) has a genome size of 36 kb. It is difficult to shuffle
this large
genome in vitro without creating an excessive number of changes which may
cause a high
percentage of nonviable recombinant variants. To minimize this problem and
achieve whole
genome shuffling of AdS, an adenovirus-phagemid was constructed. The
invention's Ad-
1 o phagemid has been demonstrated to accept inserts as large as 15 and 24
kilobases and to
effectively generate ssDNA of that size. In a further embodiment, larger DNA
inserts, as
large as 50 to 100 kb are inserted into the Ad-phagemid of the invention; with
generation of
full length ssDNA corresponding to those large inserts. Generation of such
large ssDNA
fragments provides a means to evolve, i. e. modify by the recursive
recombination methods of
the invention, entire viral genomes. Thus, this invention provides for the
first time a unique
phagemid system capable of cloning large DNA inserts (>10 KB) and generating
ssDNA in
vitro and in vivo corresponding to those large inserts.
The genomes of related serotypes of human adenovirus are shuffled in vivo
using this unique phagmid system, as described in Example 4 and illustrated in
Figure 6. The
2 o genomic DNA is first cloned into a phagemid vector, and the resulting
piasmid, designated a
"Admid," can be used to produce single-stranded (ss) Admid phage by using a
helper M 13
phage. To achieve in vivo recombination, ssAdmid phages containing the genome
of
homologous human adenoviruses are used to perform high multiplicity of
infection (MOI) on
F+ mutS E. coli cells. The ssDNA is a better substrate for recombination
enzymes such as
2 5 RecA. The high MOI ensures that the probability of having multiple cross-
overs between
copies of the infecting ssAdmid DNA is high. The shuffled adenovirus genome is
generated
by purification of the double stranded Admid DNA from the infected cells and
is introduction
into a permissive human cell line to produce the adenovirus library. This
genomic shuffling
strategy is useful for creation of recombinant adenovirus variants with
changes in multiple
3 o genes. This allows screening or selection of recombinant variant
phenotypes resulting from
combinations of variations in multiple genes.
29

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
(31 Adeno-Associated Virus (AAV~
AAV is a small, simple, nonautonomous virus containing linear single-
stranded DNA. See Muzycka, Current Topics Microbiol. Immunol. 158, 97-129
(1992). The
virus requires co-infection with adenovirus or certain other viruses in order
to replicate. AAV
is widespread in the human population, as evidenced by antibodies to the
virus, but it is not
associated with any known disease. AAV genome organization is straightforward,
comprising only two genes: rep and cap. The termini of the genome comprises
terminal
repeats (ITR) sequences of about 145 nucleotides.
AAV-based vectors typically contain only the ITR sequences flanking the
transcription unit of interest. The length of the vector DNA cannot greatly
exceed the viral
genome length of 4680 nucleotides. Currently, growth of AAV vectors is
cumbersome and
involves introducing into the host cell not only the vector itself but also a
plasmid encoding
rep and cap to provide helper functions. The helper plasmid lacks ITRs and
consequently
cannot replicate and package. In addition, helper virus such as adenovirus is
often required.
The potential advantage of AAV vectors is that they appear capable of long-
term expression
in nondividing cells, possibly, though not necessarily, because the viral DNA
integrates. The
vectors are structurally simple, and they may therefore provoke less of a host-
cell response
than adenovirus. A major limitation at present is that AAV vectors are
extremely difficult to
grow in large amounts.
B. Non-Viraf Gene Transfer Methods
Nonviral nucleic acid vectors used in gene therapy include plasmids, RNAs,
antisense oligonucleotides (e.g., methylphosphonate or phosphorothiolate),
polyamide nucleic
acids, and yeast artificial chromosomes (YACs). Such vectors typically include
an expression
cassette for expressing a protein or RNA. The promoter in such an expression
cassette can be
2 5 constitutive, cell type-specific, stage-specific, and/or modulatable
(e.g., by hormones such as
glucocorticoids; MMTV promoter). Transcription can be increased by inserting
an enhancer
sequence into the vector. Enhancers are cis-acting sequences of between 10 to
300 base pairs
that increase transcription by a promoter. Enhancers can effectively increase
transcription
when either 5' or 3' to the transcription unit. They are also effective if
located within an
3 0 intron or within the coding sequence itself. Typically, viral enhancers
are used, including
SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus
enhancers.

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
Enhancer sequences from mammalian systems are also commonly used, such as the
mouse
immunoglobulin heavy chain enhancer.
Gene therapy vectors of all kinds can also include a selectable marker gene.
Examples of suitable markers include, the dihydrofolate reductase gene (DHFR),
the
thymidine kinase gene (TK), or prokaryotic genes conferring drug resistance,
gpt (xanthine-
guanine phosphoribosyltransferase, which can be selected for with mycophenolic
acid; neo
(neomycin phosphotransferase), which can be selected for with 6418,
hygromycin, or
puromycin; and DHFR (dihydrofolate reductase), which can be selected for with
methotrexate
(Mulligan & Berg, Proc. Natl. Acad. Sci. (U.S.A.) 78, 2072 (1981); Southern &
Berg, J. Mol.
1 o Appl. Genet. 1, 327 ( 1982)).
Before integration, the vector has to cross many barriers which can result in
only a very minor fraction of the DNA ever being expressed. Limitations to
high level gene
expression include: loss of vector due to nucleases present in blood and
tissues; inefficient
entry of DNA into a cell; inefficient entry of DNA into the nucleus of the
cell and preference
of DNA for other compartments; lack of DNA stability in the nucleus (factor
limiting nuclear
stability may differ from those affecting other cellular and extracellular
compartments),
efficiency of integration into the chromosome; and site of integration.
These potential losses of efficiency can be addressed by including additional
sequences in a nonviral vector besides the expression cassette from which the
product
2 0 effecting therapy is to be expressed. The additional sequences can have
roles in conferring
stability both outside and within a cell, mediating entry into a cell,
mediating entry into the
nucleus of a cell and mediating integration within nuclear DNA. For example,
aptamer-like
DNA structures, or other protein binding sites can be used to mediate binding
of a vector to
cell surface receptors or to serum proteins that bind to a receptor thereby
increasing the
2 5 efficiency of DNA transfer into the cell.
Other DNA sequences can directly or indirectly result in avoidance of certain
compartments and preference for other compartments, from which escape or entry
into the
nucleus is more efficient. Other DNA sites and structures directly or
indirectly bind to
receptors in the nuclear membrane or to other proteins that go into the
nucleus, thereby
3 0 facilitating nuclear uptake of a vector. Other DNA sequences directly or
indirectly affect the
efficiency of integration. For integration by homologous recombination,
important factors are
the degree and length of homology to chromosomal sequences, as well as the
frequency of
31

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
such sequences in the genome (e.g., alu repeats). The specific sequence
mediating
homologous recombination is also important, since integration occurs much more
easily in
transcriptionally active DNA. Methods and materials for constructing
homologous targeting
constructs are described by e.g., Mansour (1988) Nature 336:348; Bradley
(1992)
BiolTechnology 10:534.
For nonhomologous, illegitimate and site-specific recombination,
recombination is mediated by specific sites on the therapy vector which
interact with cell
encoded recombination proteins, e.g., Cre/Lox and Flp/Frt systems, as
discussed above for in
vitro systems. See also Baubonis ( 1993) Nucleic Acids Res. 21:2025-2029,
which reports that
a vector including a LoxP site becomes integrated at a LoxP site in
chromosomal DNA in the
presence of Cre recombinase enzyme.
Nonviral vectors encoding products useful in gene therapy can be introduced
into an animal by means such as iipofection, biolistics, virosomes, liposomes,
immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial
virions, agent-
enhanced uptake of DNA, ex vivo transduction. Lipofection is described in
e.g., US
5,049,386, US 4,946,787; and US 4,897,355) and lipofection reagents are sold
commercially
(e.g., TransfectamTM and LipofectinT"'). Cationic and neutral lipids that are
suitable for
efficient receptor-recognition lipofection of polynucleotides inciude those of
Felgner, WO
91/17424, WO 91/16024.
2 0 Unlike existing viral-based gene therapy vectors which can only
incorporate a
relatively small non-viral polynucleotide sequence into the viral genome
because of size
limitations for packaging virion particles, naked DNA or lipofection complexes
can be used
to transfer large (e.g., SO-5,000 kb) exogenous polynucleotides into cells.
This property of
nonviral vectors is particularly advantageous since many genes which can be
delivered by
2 5 therapy span over 100 kilobases (e.g., amyloid precursor protein (APP)
gene, Huntington's
chorea gene) and large homologous targeting constructs or transgenes can be
required for
efficient integration. Optionally, such large genes can be delivered to target
cells as two or
more fragments and reconstructed by homologous recombination within a cell
(see WO
92/03917).
3 0 C. Applications of Gene Therag~r
Gene therapy vectors can be delivered in vivo by administration to an
individual patient, typically by systemic administration (e.g., intravenous,
intraperitoneal,
32

CA 02268265 1999-03-24
WO 98/13485 PCT/US97117302
intramuscular, subdermal, or intracranial infusion) or topical application.
Alternatively,
vectors can be delivered to cells ex vivo, such as cells explanted from an
individual patient
(e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor
hematopoietic
stem cells, followed by reimplantation of the cells into a patient, usually
after selection for
cells which have incorporated the vector.
An important application is the treatment of congenital disease, particularly
in
patients lacking both wildtype alleles of a recessive gene. The vector
introduces a wildtype
allele of the gene that allows synthesis of the corresponding gene product
compensating for
the absence of this product in the patient. Examples of recessive diseases
include sickle cell
anemia, beta-thalassemia, phenylketonuria, galactosemia, Wilson's disease,
hemochromatosis,
severe combined immunodeficiency, alpha-1-antitrypsin deficiency, albinism,
alkaptonuria,
lysosomal storage diseases, Ehlers-Danlos syndrome, hemophilia,
agammaglobuiimenia,
diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich
syndrome,
Fabry's disease and fragile X-syndrome.
Another application of gene therapy is to introduce a gene that increases the
resistance of a cell to infection by pathogenic organisms. The gene can encode
an antisense
RNA to a sequence in the microorganism not found in the patient's genome.
Alternatively,
the gene can encode a protein inhibitory to the microorganism. Examples of
microorganisms
that can be inhibited by gene therapy include viral diseases (e.g., hepatitis
(A, B, or C), herpes
virus (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and EBV), HIV, adenovirus,
influenza
virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus,
respiratory syncytial
virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus,
vaccinia virus, HTLV
virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies
virus, JC virus and
arboviral encephalitis virus) and pathogenic bacteria (e.g., chlamydia,
rickettsial bacteria,
2 S mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and
Gonococci,
klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria,
salmonella, bacilli, cholera,
tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease
bacteria). For example,
the HIV sequences Tat and Rev (Malim et al., Nature 338, 254 (1989)) are
suitable targets for
antisense RNAs or RNA binding proteins.
3 0 A further application of gene therapy in the delivery of drug resistance
genes
(polynucleotides conferring resistance to chemotherapeutic agents) to
noncancerous cells in a
patient with a view to increasing selective toxicity of the drug for cancer
cells in the patient.
33

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
For example, polynucleotides conferring resistance to a chemotherapeutic agent
(e.g., an
expression cassette driving constitutive expression of the hALDH I or hALDH 2
gene can
confer resistance to cyclophosphamide) can be transferred to non-neoplastic
cells, especially
hematopoietic cells. Other polynucleotides confernng resistance to
chemotherapeutic agents
include the cDNAs for ATP Binding Cassette transporters such as MDR1, MRPI,
cMOAT,
MRP3, MRP4, MRPS (see EP-A-96200460 filed February 22 1996).
A further application of gene therapy is to infect CD34+ cells containing the
hematopoietic stem cell and select for those cells expressing a drug
resistance gene such as
MDR1,MRPI,cMOAT, MRP3, MRP4, MRPS.
1 o In another application, gene therapy vectors are used to deliver a
negative
selection gene to cells of a patient for which selective elimination is
desired (e.g., cancer cells
or cells of a pathogen). Examples of negative selection genes include ricin or
diphtheria
toxin, and HSV thymidine kinase (tk). Vectors bearing such genes can be
selectively
introduced into target cells via a cell surface receptor for which the vector
has specific
affinity. Expression of the negative selection gene (in the case of HSV tk in
the presence of
ganciclovir) kills cells bearing the gene.
In another application, gene therapy vectors can be used as vaccines to confer
protection in subjects at risk of infection or to treat subjects who have
already been infected.
Such vectors encode immunogenic epitope(s) of pathogenic microorganisms and
express the
2 0 epitopes in the patient, particularly in target tissues at primary risk of
infection, such as the
oral and genital mucosa.
IIL Applications of Recursive Sequence Recombination to Gene Thera~v
The methods of the invention can be used to develop or improve on methods
2 5 and materials used in gene therapy, including animals, cells and vectors
for use in in vivo, ex
vivo and in vitro systems. This section discusses the application of recursive
sequence
recombination to some specific goals in gene therapy. Many of these goals
relate to
improvements in vectors used in gene therapy. Unless otherwise indicated the
methods are
applicable to both viral and nonviral vectors.
34

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
(A~ Improved Titer of a Viral Vector
In one embodiment, viruses with improved titers can be developed using the
recursive recombination methods of the invention. The property of high viral
titer can be an
advantage in propagating large amounts of a virus in vitro for use as an agent
in gene therapy.
This property is also useful if it is desired that the virus replicate in a
host tissue, such that
progeny viruses infect cells surrounding the initially infected cell. Titer of
a virus can be
improved by recursive sequence recombination. The initial substrates for
recombination can
be viral genomes showing sequence divergence as a result of natural or induced
variation.
The substrates can be whole genomes or fragments thereof. Recombination of
fragments is
useful for large genomes or in situations in which a part of the viral genome
is known to be
particularly important in conferring high titer. The substrates can be
recombined in vitro or
can be introduced into cells and recombined in vivo. Recombination in vivo can
be used to
generate progeny viruses that can be screened directly. However, recombination
in vitro
leads to recombinant genomes or fragments thereof. Whole recombinant genomes
can be
packaged into viruses using a packaging cell line or an in vitro packaging
system. Fragments
of genomes are usually first assembled by DNA ligation. They are subsequently
inserted into
a viral genome before packaging. Irrespective of the precise route, one
arrives at a population
of viruses having genomes at least part of which constitutes a recombinant
segment.
The collection of viruses with recombinant genomes can be screened simply
2 0 by propagating the viruses in cell culture for several generations. The
viruses with the highest
titer thereby acquire the highest representation among progeny viruses. If
desired, viruses can
be plaque-purified and titers of individual viruses compared to identify the
very best titer of
viruses from a round of recombination. Alternatively, the viruses can be
purified by serial
dilution to determine the very best titer viruses from a round of
recombination.
The genomes from viruses surviving screening are subject to a further round of
recombination, which again can be performed in vivo or in vitro. For in vivo
recombination,
viruses having genomes containing the recombinant segments can, for example,
be infected
into a cell at high multiplicity. For in vitro recombination, viral DNA is
isolated from viruses
harboring recombinant DNA. The genomes from viruses surviving screening can be
3 0 recombined with each other or with fresh substrates obtained from similar
sources to the
initial substrates. In some recombination steps, it is desirable to include an
excess of

CA 02268265 1999-03-24
WO 98/13485 PCT/C1S97/17302
wildtype version of the viral genome to reduce silent mutations. Again,
recombination can be
performed with whole genomes or fragments thereof. Selection is repeated as
before.
After several rounds of recombination and selection, viral mutants, or clones,
capable of producing the desirable titer can be obtained. For example, without
concentration
of an infected cell culture, it is possible to achieve a concentration of
evolved virus of at least
106, lOx or 10'° viruses/ml.
(B,~proved Infectivity of a Virus
The infectivity of a virus means the percentage of viruses that infect a cell
when an inoculum of viruses is contacted with an excess of cells. Obtaining a
high infectivity
1 o is particularly important with respect to the intended target cell-type.
Thus, if a viral vector is
being used to deliver a beneficial expression product to a target tissue
(e.g., lung cells lacking
a functional endogenous CFTR gene), it is usually desirable that as high a
percentage of
viruses as possible infect that cell type.
The selection of substrates and means of recombination follows the same
principles as discussed for improved viral titer. However, the means of
screening viruses
bearing recombinant genomes is usually different. The previous selection does
not
necessarily select for viruses having high infectivity because high titer can
also be conferred
by high burst size per cell. To screen more specifically for high infectivity,
clonal isolates of
viruses bearing recombinant segment are used to infect separate cultures of
cells. The
2 0 percentage of viruses infecting cells can then be determined by, for
example, counting cells
expressing a marker expressed by the viruses in the course of infection. After
several rounds
of recombination and screening, viruses harboring recombinant genomes capable
of infecting
50, 75, 95 or 99% of target cells are obtained.
(Cl Improved Packaging Capacity of a Virus
Viruses and vectors with the capability of incorporating increasing amounts of
recombinant nucleic acids sequences, such as having an improved packaging
capacity within
the viral capsid, can be developed using the recursive recombination methods
of the
invention. As noted above, the viruses commonly used in gene therapy can
package only a
limited genome length, thus, restricting the capacity of viruses to
accommodate large inserts.
3 0 Capacity of a virus can be improved using similar principles to those
discussed above. In
these methods, the viral genome to be lengthened should have a site into which
increasing
lengths of nucleic acid can be inserted in successive rounds of screening
without affecting
36

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
other viral functions. Initially, one can start with a viral genome having an
insert such that
the combined length of the genome is close to the existing maximum capacity of
the virus.
The initial substrates for recombination are variant viral genomes as in the
other methods.
The variation usually occurs other than in the length-conferring insert
because the insert is
replaced in actual use of the vector. One source of starting substrates can be
viral genomes
known to show sequence similarity with the virus to be evolved but which have
a larger
genome packaging capacity. Recombination proceeds in the same manner as
discussed
above. Viruses having recombinant genomes are then screened for titer or
infectivity as
discussed above. Recombinant genomes from viruses having the best titer and/or
infectivity
are manipulated to introduce a further insert to increase the genome length.
There follow
further cycles of recombination, screening and increasing genome length, until
viruses are
achieved that can accommodate inserts of the desired size. For example, the
maximum insert
size used in most existing adenoassociated viral vectors is about 5 kb, which
can be increased
to 10, 15, 20 or 50 kb or more.
(D) Improved Stability of a Virus
Viruses with improved stability can be developed using the recursive
recombination methods of the invention. Stability of a virus for use in gene
therapy is
important both in prolonging the shelf life of the virus as a drug between
manufacture and
administration, and in the subsequent ability of the virus to resist cellular
degradative
2 0 mechanisms before reaching its target. The principles for selection of
starting substrates and
performing recombination are the same as in other methods described above.
Viruses bearing
recombinant genomes that have evolved to acquire greater stability can be
selected by
exposing the viruses to destabilizing conditions and recovering surviving
viruses. For
example, destabilizing conditions include temperature (hot or cold),
mechanical disruption
2 5 (e.g., centrifugation or sonication), exposure to chemicals or exposure to
biological degrading
agents such as proteases (e.g. serum proteases). Viruses surviving exposure to
destabilizing
conditions are identified by propagation of treated viruses and collection of
progeny.
Sometimes, propagation proceeds only for one or a limited number of
generations, since
otherwise progeny viruses become biased toward those having genomes favoring
high titer in
3 0 addition to those having genomes conferring stability.
37

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
lEl Improved Expression or Expression Regulation of a Vector Coded Seauence
Improved expression of a gene sequence of interest can be achieved by
performing the recursive sequence recombination methods of the invention.
Usually viral or
nonviral vectors used in gene therapy encode a product to be expressed in an
intended target
cell. The product can be a protein or RNA. such as an antisense RNA or RNA
that
specifically binds a target protein, i.e., an aptamer. Usually, the coding
sequence is operably
linked to an additional sequence, such as a regulatory sequence, to ensure its
expression, such
as some or all of the following: an enhancer, a promoter, a signal peptide
sequence, an intron
and/or a polyadenylation sequence. A desirable goal is to increase the level
of expression of
1 o functional expression product relative to that achieved with conventional
vectors. Expression
can effectively be improved by a variety of means, including increasing the
rate of production
of an expression product, decreasing the rate of degradation of the expression
product or
improving the capacity of the expression product to perform its intended
function.
Improvement of the latter four parameters for drug transporters including but
not limited to
M1JR1, MRPI, cMOAT, MRP3, MRP4, MRPS, an embodiment of this invention, results
in
preferred variants of these transporters. These are applied in protective gene
therapy of a
wide variety of tissues including but not limited to bone marrow, kidney,
Iiver, intestine and
heart. These improved drug transporter variants are also applied in dual
vectors such as dual
retroviral vectors which carry the transporter variant and a gene encoding a
therapeutic gene
2 o such as the gene for lysosomal glucocerebrosidase deficient in Gaucher
disease. In vivo
selection for the improved drug transporter variant present on the dual
construct results in
selection for the therapeutic sequence as well and thus has therapeutic
benefit.
Improved expression of selection markers can be achieved by performing
recursive sequence recombination. For purposes of selection, a gene product
expressed from
a vector is sometimes an easily detected marker rather than a product having
an actual
therapeutic purpose, e.g., a green f3uorescent protein (see Crameri (1996)
Nature Biotech.
14:315-319) or a cell surface protein. However, some genes having a
therapeutic purpose,
e.g., drug resistance genes, themselves provide a selectable marker, and no
additional or
substitute marker is required. Alternatively, the gene product can be a fusion
protein
3 o comprising any combination of detection and selection markers.
The substrates for recombination can be the full-length vectors or fragments
thereof including coding sequence and/or regulatory sequences to which the
coding sequence
38

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
is operably linked. The substrates can include variants of any of the
regulatory and/or coding
sequences) present in the vector. If recombination is effected at the level of
fragments, the
recombinant segments should be reinserted into vectors before screening. If
recombination
proceeds in vitro, vectors containing the recombinant segments are usually
introduced into
cells before screening. Cells containing the recombinant segments can be
screened by
detecting expression of the gene encoded by the selection marker. Internal
reference marker
genes can be included on the vector to detect and compensate for variations in
copy number
or insertion site. For example, if this marker is green fluorescent protein,
cells with the
highest expression levels can be identified by FACSTM. If the marker is a cell
surface protein,
such as MDRI or cMOAT, the cells are stained with a reagent having affinity
for the protein,
such as antibody, and again analyzed by FACSTM. Recombinant segments from the
cells
showing highest expression are used as some or all of the substrates in the
next round of
screening.
Evolution of Cytomegalovirus Transcriptional Regulatory Elements
The major immediate-early (IE} region transcriptional regulatory elements,
including promoter and enhancer sequences (the promoter/enhancer region), of
cytomegalovirus (CMV) is widely used for regulating transcription in vectors
used for gene
therapy because it is highly active in a broad range of cell types. Optimized
CMV
transcriptional regulatory elements which direct increased levels of transgene
expression is
2 0 generated by the recursive recombination methods of the invention,
resulting in improved
efficacy of gene therapy. As the CMV promoter and enhancer is active in human
and animal
cells, the improved CMV promoter/enhancer elements are used to express foreign
genes both
in animal models and in clinical applications.
A library of chimeric transcriptional regulatory elements is created by DNA
2 5 shuffling of wild-type sequences from five related strains of CMV. The
promoter, enhancer
and first intron sequences of the IE region are obtained by PCR from the CMV
strains: human
VR-538 strain AD169 (Rowe (1956) Proc. Soc. Exp. Biol. Med. 92:418; human V-
977 strain
Towne (Plotkin (1975) Infect. Immunol. 12:521-527); rhesus VR-677 strain 68-1
(Asher
{1969) Bacteriol. Proc. 269:91); vervet VR-706 strain CSG (Black (1963) Proc.
Soc. Exp.
3 0 Biol. Med. I 12:601 ); and, squirrel monkey VR-1398 strain SqSHV (Rangan (
1980) Lab.
Animal Sci. 30:532). The promoter/ enhancer sequences of the human CMV strains
are 95%
homologous, and share 70% homology with the sequences of the monkey isolates,
allowing
39

CA 02268265 1999-03-24
WO 98/13485 PCT/i7S97/17302
the use of family shuffling to generate a library great diversity. Following
shuffling, the
library is cloned into a plasmid backbone and used to direct transcription of
a marker gene in
mammalian cells. An internal marker under the control of a native promoter can
be included
in the plasmid vector. Expression markers, such as green fluorescent protein
(GFP) and
S CD86 (also known as B7.2, see Freeman (1993) J. Exp. Med. 178:2185, Chen
(1994) J.
Immunol. 152:4929) can also be used. In addition, transfection of SV40 T
antigen-
transformed cells can be used to amplify a vector which contains an SV40
origin of
replication. The transfected cells are screened by FACS sorting to identify
those which
express high levels of the marker gene, normalized against the internal marker
to account for
differences in vector copy numbers per cell. If desired, vectors carrying
optimal, recursively
recombined promoter sequences are recovered and subjected to further cycles of
shuffling and
selection.
(F) Improved Expression and/or Function of Drug Resistance Sequences
The recursive recombination methods of the invention also provide for means
to improve the expression of drug resistance sequences/ proteins. Many
treatment regimes
entail administration of drugs having side-effects on a particular cell type
in the body. For
example, chemotherapy is notorious for killing cells other than the targeted
cancer cells. See
Licht (1995) Cytokines & Molecular Therapy 1:11-20. Myelosuppression, or bone
marrow
toxicity, is dose-limiting for many chemotherapeutic agents. This is not only
a dangerous
2 0 side effect but also limits the effectiveness of chemotherapy. Indeed, the
chemotherapy can
be fatal, either directly by loss of blood cell function or indirectly by
causing secondary
cancers such as leukemia. It is possible to protect hematopoietic cells by
delivering drug
resistance proteins via gene therapy. This principle has been demonstrated by
a number of
studies in which murine bone marrow cells were protected against
chemotherapeutic
2 5 alkylating agents by the overexpression of a protective alkyltransferase.
Other drug resistance
proteins can be used for chemoprotection of normal tissues and can be targets
for improved
expression using the methods of the invention. They include, for example,
glutathione-S-
transferase, dihydrofolate reductase and superoxide dismutase.
Alkylating agents are especially toxic to the hematopoietic system, with
3 0 myelosuppression being the dose-limiting side effect. Hematopoietic cells
are so susceptible
to alkylating agents that iatrogenic leukemias are a common occurrence.
Alkylation therapy
can also cause severe pulmonary toxicity and result in dose limitations.
Examples of other

CA 02268265 1999-03-24
WO 98/13485 PCTIUS97/17302
drugs that have dose limitations due to toxicity to vital organs are etoposide
(e.g. kidney),
cisplatin (e.g. kidney), taxol (e.g. lung), anthracyclines (e.g. heart), See
Perry et al, The
Chemotherapy Source Book,1991, Williams and Wilkins, Baltimore, USA, ISBN
0-683-06859-08. This limitation sensitivity can be attributed to the low
expression of the
DNA repair protein O6-methylguanine-DNA methyltransferase in hematopoietic
cells (also
called O6-alkylguanine-DNA alkyltransferase, MGMT or alkyltransferase; EC
2.1.1.63).
Alkylating agents, especially nitrosoureas, as used either alone or in
combination with other
drugs to treat many types of cancer, such as Hodgkin's and non-Hodgkins
lymphomas,
multiple myeloma, malignant melanoma, brain neoplasms, gastrointestinal
cancers and lung
cancers. Together these cases constitute over one third of all cancers
diagnosed. Thus,
improving the effectiveness and decreasing the toxicity of alkylation-based
chemotherapeutic
regimens will have a profound impact on health care.
The introduction of drug-resistance genes into bone marrow stem cells or
pulmonary cells or kidney cells or heart cells or liver cells or intestinal
cells via gene therapy
is one way to overcome the limitations of chemotherapeutic regimens. In the
case of bone
marrow, one strategy is to transduce the cells ex vivo with the drug
resistance gene and
repopulate the bone marrow with these cells before or after chemotherapy. Bone
marrow is a
relatively easy tissue to extract, manipulate and reintroduce into the body.
Kidney or liver or
heart or intestine or central nervous tissue or other tissues are protected by
retrovirus or
2 0 adenovirus or AAV vectors or nonviral vectors carrying drug resistance
genes after in vivo
administration of the recombinant adenovirus into the patient and targeting of
the virus to the
desired tissue followed by chemotherapy aimed at the killing of a tumor in a
tissue other than
the protected tissue.
MGMT is found in all organisms examined, prevents the mutagenic, cytotoxic,
2 5 and carcinogenic effects of chemotherapeutic alkylating agents. MGMT
removes alkyl
groups attached by such chemicals from the O6 position of guanine. These alkyl
groups are
transferred irreversibly to a cysteine in the active site of the MGMT protein,
inactivating the
alkyltransferase. Thus, the enzyme is a suicide enzyme and can act only
stoichiometrically,
which is an important barrier to improvement of MGMT. Because each protein
module acts
3 0 only once in a suicidal manner, the protection afforded a cell is
determined not only by the
activity (quality) of the MGMT but also by the number of MGMT molecules.
Cells, such as
bone marrow cells, which express little or no alkyltransferase are very
sensitive to laboratory
41

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Day
(1980) Nature
288:724-727) and clinically used nitrosoureas (Erickson ( 1980) Nature 288:727-
729). Thus,
myelosuppression is a serious problem with drug-based chemotherapeutic
regimens (DeVita
(1993) Cancer: Principles and Practice of Oncology), but it has been overcome
in
experiments in which the wild-type human, mouse, or bacterial alkyltransferase
genes were
transduced into human and mouse hematopoietic cells. The overexpressed genes,
carried on
retroviral vectors, protected stem cells in culture from killing by
nitrosoureas (Allay (1995)
Blood 85:3342-3351; Moritz (1995) Cancer Res.55:2608-2614). Furthermore, when
these
cells were transplanted into the bone marrow of mice, the protection proved to
be long-lasting
in vivo (Maze (1996) Proc. Natl. Acad. .Sci. USA 93:206-210). Similar effects
were seen
when liver and thymus rather than bone marrow were targeted (Dumenco (1993)
Science
259:219-222; and Nakatsuru ( 1993) Proc. Natl. Acad Sci. USA 90:6468-6472).
This protective effect of MGMT can be improved by recursive sequence
recombination in several respects. First, novel variants can be selected
having higher specific
activity, i.e., faster repair of cytotoxic alkylation-induced lesions. Thus,
for a given
expression level, bone marrow cells will be better protected. Some improvement
in MGMT
has been reported (Christians ( I 996) Proc. Natl. Acad. Sci. . USA 93:6124-
6128) using a
conventional cassette mutagenesis. Second, novel variants can be selected for
resistance to
inhibitors of wild-type alkyltransferases, such as O6-benzylguanine. Such
inhibitors are
2 0 sometimes used to suppress endogenous alkyltransferases present in cancer
cells (Pegg { 1995)
Progress in Nucleic Acid Res. and Molec. Biol. 51:167-223). Inhibitor-
resistant MGMT can
be used to transfect bone marrow in treatment protocois in which alkylating
agents are
combined with inhibitors of alkyltransferases. Third, novel variants of the
coding sequence
and/or operably linked regulatory sequences can be selected for improved
expression of
MGMT. Fourth, variants of MGMT can be produced that bind to but do not remove
alkyl
adducts from DNA, effectively resulting in DNA-protein crosslinks more toxic
to the cell
than the alkyl adducts alone. Vectors expressing the mutant variants can be
targeted to cancer
cells before treatment with the alkylating substrate. Fifth, MGMT variants can
be selected to
protect mammalian cells against the clinically relevant nitrosoureas. For this
purpose,
3 o selection should be preferably performed in mammalian cells rather than
bacterial cells,
because the protective effect of MGMT against nitrosoureas is stronger in the
former.
42

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
The sometimes-low transfection efficiencies of gene therapy are not a major
limitation in ex vivo methods because alkylation treatment effectively serves
as a positive
selection for transfected cells. In contrast, low transfection efficiencies
can be a problem in in
vivo gene replacement therapy because there is no generally positive
selection, only negative
selection by tumoricidal gene therapy. Improved means of positive selection
for in vivo gene
replacement therapy allows, for example, a relatively small number of
chemoresistant
hematopoietic cells to repopulate the bone marrow.
A drug-resistance gene is a starting material for improvement using the
methods of the invention is the mufti-drug resistance gene MDR-1. MDR-1
encodes a plasma
membrane glycoprotein called "P-glycoprotein (Pgp") which acts as an ATP-
dependent drug
efflux pump and confers chemoresistance to a wide variety of drugs (Chin
(1993) Adv.
Cancer Res. 60:157). Cells not expressing MDR-1 are exquisitely sensitive to
drugs such as
vincristine, etoposide, and colchicine. This same chemoresistance property,
which when
expressed by tumor cells can frustrate chemotherapy efforts, can be turned to
an advantage
when used as a positive selectable marker. Metz (1996) Lirology 217:230-241,
reported a
20-fold higher stringency when selecting for MDR1 expression compared to neo
selection.
P-glycoprotein has been demonstrated to positively select for transformed
cells in the irz vitro
correction of cells from at least two different genetic diseases, Fabry
disease (Sugimoto
(1995) Human Gene Therapy 6:905-915) and chronic granulomatous disease
(Sokolic (1996)
2 o Blood 87:42-50). However, there is no reason to believe that nature has
optimized MDR1 for
activity against man-made drugs. Improving MDR-1 by recursive recombination to
improve
protection of cells from drugs such as etoposide and colchicine will allow the
use of higher
levels of such selective agents, which will increase the selection stringency
and better
differentiate between transformed and non-transformed cells.
MDR-1 is improved/modified by DNA shuffling followed by positive selection
in mammalian cells. Randomly mutated pools of MDR-1 are inserted into
appropriate vectors
(e.g., retroviral, adenoviral vectors) and transformed into drug-sensitive
cells. Selection with
colchicine and/or etoposide and/or vincristine will identify active MDR-1
variants. The
MDR-1 genes are rescued from surviving cells and subjected to additional
rounds of
3 o recombination and selection with increasing doses of drugs.
Because some mammalian cells already express high levels of P-glycoprotein,
it might be difficult to determine whether the improved MDR-1 transgene is
expressed in
43

CA 02268265 1999-03-24
WO 98/13485 PCTIUS97/I7302
these cells; i.e., the background will be high. In this case the endogenous P-
glycoprotein is
inactivated with a well-characterized inhibitor such as verapamil, and
transform with a
marker MDR-1 transgene that encodes a mutant P-glycoprotein resistant to the
inhibitor yet
highly active against the cytotoxic drug. Such a variant is created by
selecting MDR-1 mutant
pools in the presence of both the inhibitor and the cytotoxic drug(s), such as
colchicine. For
example, the methods of the invention are used to create alkyltransferase
mutants super-active
against the cytotoxic chemical N-methyl- N-nitro- N-nitrosoguanidine (MNNG)
and
completely resistant to the alkyltransferase inhibitor benzylguanine.
MDR-1 thus optimized as a positive selection marker is inserted into the
vector
of choice. The vector can also be optimized by DNA shuffling, either by itself
or in
combination with MDR 1 mutagenesis (MDR 1 and the vector shuffled as a unit).
Shuffling
the entire construct allows many parameters to be tested at once. Bicistronic
arrays, 2 genes
transcribed as one mRNA from the same promoter but translated from separate
ribosome
binding sites, can be used (Sugimoto ( 1995) Human Gene Ther., supra).
Shuffling the entire
array or the whole construct can be used to optimize secondary structure of
the bicistronic
mRNA to improve translation of the second, downstream gene. For example, a
bicistronic
retroviral vector encoding MDR 1 and a gene complementing a genetic defect can
be
constructed and optimized using the methods of the invention. The entire
vector can be
mutagenized by DNA shuffling and reassembled. Additionally, the vector can be
packaged as
2 0 virus by a packaging cell line, transfected into the defective cells, and
selected with
colchicine. Selection is effected by analyzing surviving cells for
complementation of the
genetic defect.
Further candidates for improvement are members of the ATP Binding Cassette
(ABC) family of transporters. Members of this family include but are not
limited to MDR1,
2 5 MDR2, MRP 1. MDR 1 and MRP 1 encode ATP dependent drug efflux pumps useful
for the
protection of stem cells in an ex vivo gene therapy setting. Other ABC
transporters include
the canalicular Multispecific Organic Anion Transporter (cMOAT), MRP3, MRP4
and MRPS
subject of patent application EP-A-96200460 (filed February 22 1996). cMOAT is
involved
in the transport of organic anions such as glutathione and glucuronide
conjugates of
3 o cis-platinum and etoposide of which the parent compounds are used in
cancer treatment
regimens (Paulusma (1996) Science 271:1 i26-1128). Desired chemotherapeutic
agents such
as etoposide and mitoxantrones do not represent good substrates for MDR1 or
cMOAT but
44

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
are drugs that are clinically very desirable agents and therefore mutant
versions transporting
etoposide, mitoxantrones or cisplatin with high efficiency are useful for
protective gene
therapies including gene therapies using MDRI and cMOAT.
A drug transporter gene can be evolved/modified not only to confer improved
protection to drugs it already recognizes (e.g., etoposide) but also to confer
protection against
drugs not recognized by wildtype MDR-1, such as alkylating agents. For
example, an MDR-1
gene can be modified by recursive recombination (evolved) to pump alkylating
agent out of a
cell, thus serving as a complement to MGMT (described above). For example,
both the
MGMT and MDR-1 genes can be transduced into stem cells before combination
1 o chemotherapy in which one of the drugs is an alkylator. Studies in which
stem cells were
transduced with the wild-type MDR-1 gene gave results similar to those cited
above with
MGMT for alkylating agents (Sorrentino ( 1992) Science 257:99).
Another suitable gene for evolution/ modification using the methods of the
invention is glutathione-S-transferase, which detoxifies alkylating drugs in
the cytoplasm,
complementing MGMT. It acts on drugs after they have entered the nucleus and
damaged
DNA. Some improvements in glutathione-S-transferase resulting from
conventional cassette
mutagenesis in bacteria have already been reported (Gulick (1995) Prod. Natl.
Acad. Sci.
USA 92:8140-8144). Further evolution by recursive sequence recombination will
provide
additional improvements. The improvement gene can then be transfected into
stem cells or
2 0 lung cells on its own or in combination with MGMT.
Other drug-resistance genes are candidates for evolution for use in
suppressing
side effects in other tissues. For example, bleomycin is an antineoplastic
whose major
toxicity is to pulmonary cells. The protein bleomycin hydrolase can protect
cells from
bleomycin, and the human gene was recently cloned (Bromme (1996) Biochemistry
35:6706-
2 5 714). The gene can be improved by gene shuffling and used to protect
pulmonary cells in
cancer patients.
Inhibition of replication and spread of infective HIV-1 by retroviruses
expressing anti-HIV molecules such as HIV specific antisense or ribozymes have
been shown
to be a promising approach for the treatment of HIV-1 infected individuals.
Such therapy can
3 0 only be expected to be successful in the long run, when virus replication
is prevented in the
majority of CD4+ (HIV-1 permissive) cells. Most of the CD4+ T-lymphocytes and
macrophages have a limited Iifespan so that transduction of these cells can
provide no lasting

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
protection. Therefore, hematopoietic stem cells are the target cells of choice
for HIV gene
therapy. Unfortunately, preciinical and clinical studies demonstrate that
after
retransplantation of transduced hemopoietic stem cells only 0.1 % of the
peripheral blood cells
contain the virus. Introduction of a gene that enables in vivo selection of
transduced cells
next to the antiviral polynucleotide sequence may overcome this problem. In
another
application MDR1 or MRP1 or cMOAT or MRP3 or MRP4 or MRPS variants are
generated
that more efficiently pump HIV inhibitors such as the clinically used reverse
transcriptase
inhibitors AZT and ddC or HIV protease inhibitors or combinations thereof.
These are
desired for use in stem cell based anti HIV gene therapy using in vivo
selection of AZT
resistant stem cells carrying an AZT transporting MDRI or cMOAT variant and an
anti-HIV
sequence such as a ribozyme or antisense sequence. Since AZT and ddC are known
for their
toxic effects on hematopoietic cells, the MDRI/AZT system provides an
efficient in vivo
selection system for stem cell-based gene therapy protocols to treat HIV
infected individuals.
In other embodiments, candidate genes for improvement include the genes
1 S encoding DNA ligase and topoisomerase to protect against ionizing
radiation (Boothman
(1994) Cancer Res. 54:4618-4626), genes encoding nucleotide excision repair
enzymes such
as T4 endonuclease V to protect against UV irradiation and skin cancer, and
genes encoding
alkaline phosphatase endonuclease and glycosylases to improve the base
excision repair
pathway which is crucial to ward off the effects of oxidative DNA lesions
thought to cause
2 0 many types of cancer and accelerated aging.
Evolution/modification of drug-resistance genes and associated regulatory
sequences using the methods of the invention falls under the general approach
discussed
above for improving gene expression. >'Iowever, in evolving drug-resistance
genes, it is
sometimes desirable not only to improve expression of the gene but to increase
the degree of
2 5 resistance conferred by the gene product with a particular drug. In this
situation, it is
preferable that substrates for recombination include the drug-resistance gene
as well as
associated regulatory sequences so that the resistance gene can be evolved
within the genetic
context in which it is to be expressed. Diversity between the initial
substrates can be the
result of induced mutations, natural drug-resistance genes from different
sources, and
3 0 mutations already known to confer improved properties.
For example, the cDNA sequences of five different mammalian species of
MGMT (human, rat, mouse, hamster, and rabbit) have been reported, and, despite
very
46

CA 02268265 1999-09-27
10
extensive homology, variations do exist, as illustrated in Figure 4. Following
is an alignment
showing the human amino acid sequence on the top line with other amino acid
sequence
found in nature shown below the human sequence.
MDKDCEMKRT TLDSPLGKLE LSGCEQGLHE IKLLGKGTSA ADAVEVPAPA 50
AET KL YS VFH AM C R G RFPSGK PN T PT A TP
I D K A I S S S K C
E A S
AVLGGPEPLM QCTAWLNAYF HQPEAIEEFP VPALHHPVFQ QESFTRQVLW 100
EL S V ET E RE A TPGL L D
G H Q S
KLLKVVKFGE VISYQQLAAL AGNPKAARAV GGAMRGNPVP ILIPCHRVVC 150
TV S IR
M N
SSGAVGNYSG GLAVKEWLLA HEGHRLGKPG LGGSSGLAGA WLKGAGATSG 200
D SI H QT IPTRQ A SKGL I S R SSFESTS
N A K C D T T P G
SPPAGRN 207 (SEQ ID N0:29)
PELS
K
The natural variations can be incorporated by any of the formats discussed in
Section II to generate recombinant forms of MGMT including natural segments
unique to
-s o human and nonhuman forms, as discussed in Example 3. For example,
oligonucleotides can
be designed to encode all the different combinations of natural variants, and
these
oligonucleotides will be mixed in with the fragmented wild-type human gene. A
surprisingly
small number of oligonucleotides (twenty-one) can be used if they are
degenerate at positions
at which more than two amino acids are represented in nature (see Figure 3).
The
3 5 oligonucleotides shown in Figure 3 contain up to twenty one bases of
nonhomology to the
human sequence flanked on either side by a 20 base sequence perfectly matched
with the
human MGMT sequence. Another use of "oligo spiking" is to bias shuffled gene
pools
toward known desirable mutations such as the V 139F mutation demonstrated to
improve the
wild-type protein (Christians (1996) Prod. Natl. Acad Sci. USA 93, 6124-6128),
or mutations
4 o conferring O6-benzylguanine resistance.
47

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
An alternative to "oligo spiking" is to obtain all the individual cDNAs and
shuffle them together. This option might have some tendency to dilute the
human character
of the pool leading to immunogenic problems when used in human gene therapy,
but this
problem can be overcome by backcrossing mutants with the wild-type human gene
to
eliminate non-useful mutations.
Recombined drug-resistance genes and vectors encoding them can readily be
screened for improved expression. Cells containing vectors containing
recombinant segments
are exposed to the drug and surviving cells recovered. These cells are
enriched for
recombinant segments conferring improved resistance to the drug. Screening can
be made
more stringent in successive rounds by increasing the concentration of drug or
duration of
exposure thereto.
The final round of selection is usually performed in stem cells because some
of
the component factor contributing to the end point of drug-resistance may be
cell-type
dependent (see Examples 5 and 6). Because expression levels are important for
the protective
effect, manipulating vector sequences other than that encoding drug resistance
genes such as
MGMT, MDR1, cMOAT, MRPI, MRP3, MRP4 and MRPS, provides an important source of
improvement. The vectors are selected based on desired endpoints, such as the
ability to
protect cells from alkylating agents. The endpoint is achieved by a variety
and a combination
of components too complicated to predict, including enhanced transduction,
better vector
2 0 stability, and improved transcription of the gene in addition to improved
or altered function of
the drug resistance gene.
(G) Evolution of Transducing Vectors for Integration and Stable Expression in
Mammalian Stem Cells
Vectors having new and/or improved ability to infect, integrate and express
2 5 themselves in hematopoietic stem cells can be developed using the
recursive recombination
methods of the invention. A major goal in gene therapy is to develop practical
methods to
efficiently integrate DNA constructs into human stem cells. A practical method
for
efficiently integrating retroviruses into stem cells allows repopulation of
patients with
autologous bone marrow that had been genetically modified with traits of
interest. For
3 0 example, the stem cells are engineered to express trans-dominant factors
that interfere with
viral replication. Stem cells are engineered to express wild type or
engineered transgenes that
complement a defined genetic defect, such as Gaucher's disease. MDR genes or
48

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
alkyltransferase genes are inserted into stem cells to confer resistance to
chemotherapeutic
agents. Gene encoding T cell receptors specific for cancer or pathogen
epitopes of interest
are inserted for expression upon maturation of the stem cell.
However, stem cells are difficult to purify and rapidly lose their pluripotent
phenotype if propagated in vitro. Retroviruses are very inefficient at
integrating into
nondividing cells in general, and stem cells in particular. Thus, recursive
recombination is
used to evolve a factor or set of factors that, upon infection with and
expression of the
retrovirus genes prior to integration, can transiently or permanently render a
stem cell
susceptible to retroviral integration while at the same time remaining
pluripotent:
In one embodiment, large (> 106) libraries of retroviruses expressing
candidate
factors for transiently perturbing stem cells so as to promote retroviral
integration are made.
Such factors include, but not be limited to: HIV matrix, HIV vpr, random
fragments of HIV
and other lentiviruses (the only class of retroviruses able to efficiently
transduce non-dividing
cells); cDNAs from stem cells; cDNA from stromal cell cultures (which make
factors that
influence the differentiation state of stem cells, and over production or
evolution of
recombined forms exert the desired effect); or, any other cytokine or growth
factor. Such
libraries are used in the in vitro and in vivo recursive recombination methods
of the invention,
as generally described above, to create a retrovirus which can efficiently
infect, integrate and
express sequences and proteins of interest in non-dividing stem cells.
2 o Another embodiment repopulates SCID or SCID/NOD mice with human stem
cells that have been transduced by a retrovirus modified by the above methods.
Progeny of
retroviruses from stem cells that were successfully transduced by a member of
the initial
retrovirus recombinant segment library are recovered. Selection markers, such
as green
fluorescent protein (GFP), drug markers, or cell sorter (FACS) markers may be
encoded in
the transducing retrovirus to facilitate recovery of repopulating stem cells
transduced with a
retrovirus construct. Sequences encoding the factors to be evolved/modified or
the entire
integrated retroviral genome can be recovered. Further rounds of recursive
sequence
recombination can be repeated until the desired efficiency/efficiency of stem
cell transduction
is achieved.
3 0 A murine SCID/NOD immunodeficient system that can be repopulated with
primitive human hematopoietic stem cells can be used (Dick (1996) CSH Gene
Therapy
abstract #11). Retroviruses can infect these stem cells with very low but
detectable
49

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
efficiency. Progenitor cells with integrated retrovirus can be recovered from
peripheral blood
cells in this SCID/NOD repopulation model. This and analogous repopulation
systems
therefore forms the basis for selecting retroviruses with improved efficiency
of integration
into primitive pluripotent cells. As noted above, including GFP in the vector
allows for
FACS purif cation of cells expressing retroviral-encoded proteins after
repopulation. If the
repopulation is initially very inefficient, a selectable gene such as Neo or
TK to selectively
culture transduced cells is also expressed.
In another embodiment, rather than removing infected stem cells and isolating
retroviral sequences for further rounds of recursive recombination, lethally
irradiated
to retroviral producing helper lines containing recombinant sequences are
injected into the
SCID/NOD bone marrow. With this technique, recursive recombination takes place
in vivo;
the stem cells remaining in the special environment of the bone marrow, an
environment that
may prove impossible to mimic in vitro.
In a further embodiment, recursive recombination is used to develop a means
15 by which viruses which cannot normally lack the means to integrate into non-
dividing cells.
This method incorporates HIV proteins which are required for HIV to integrate
into
nondividing cells, into other vectors of interest. For example, integrase, the
enzyme
responsible for mediating the integration of the viral genome in the host cell
chromosome,
can suffice to connect the HIV-1 preintegration complex with the cell nuclear
import
20 machinery. Viral matrix and Vpr proteins also play important roles in the
ability of HIV to
integrate into non-dividing cells. See Gallay (1997) Proc. Natl. Acad. Sci.
USA 94:9825-
9830. Repeated cycles of recursive recombination, as DNA shuffling, are
carried out until the
desired property is conferred to the vector or sequence of interest.
In another embodiment, before recursive recombination, long term bone
2 5 marrow cultures are stimulated to cycle in vitro. This results in
increased retroviral
transduction of the stem cells in both a murine SCID/Beige repopulation assay
(Agatsuma
(1997) Antiviral Res. 34:121-130) and in stem cell repopulation of terminal
human myeloma
patients with transduced bone marrow cells. Cycling stem cells are more
susceptible to
transduction. Thus, stem cells can be stimulated such that they are more
susceptible to
3 0 retroviral transduction and yet remain pluripotent.

CA 02268265 1999-03-24
WO 98/13485 PCT/LTS97/17302
j~-I Improved Tissue Specificity of a Vector
Vectors with new and/or improved tissue specificity (tissue tropism) can be
developed using the recursive recombination methods of the invention. In most
gene therapy
applications, it is desirable that the gene therapy vector be delivered with a
high degree of
specificity to a particular tissue type. Specificity of cellular targeting is
a key issue impacting
the safety and practicality of these vectors for in vivo gene therapy. Thus,
there is a need to
restrict and/or redirect the specificity of gene therapy vectors, such as
adenovirus.
One example illustrating the need to deliver a gene therapy vector a specific
tissue type involves delivering a wildtype CFTR gene to cystic fibrosis
patients. The CFTR
gene should be delivered mainly to pulmonary tissue. In a second example,
where the gene
therapy vector encodes a chemotherapeutic agent, it is desirable that the
agent be delivered to
neoplastic cells and not normal cells.
The strategy in selecting substrates and recombination formats is in general
similar to those discussed before. Substrates for recombination can be whole
viral genomes
or can be fragments encoding the viral proteins thought to interact with
cellular receptors. If
such fragments are recombined, the recombination products should be reinserted
into viral
genomes, and the genomes packaged to form viruses before screening.
For example, for evolution of vesicular stomatitis virus (VSV) to infect new
target cells, recursive recombination should focus on G-protein sequences,
because the G
protein is expressed on the capsid's outer surface (Schnell (1996) Proc. Natl.
Acad. Sci. USA
93:11359-11365). Furthermore, it has been technically difficult to generate
viruses encoding
the vesicular stomatitis virus G-protein (VSV-G) because it is too toxic to
the host cells to
allow for viral propagation (Yoshida (1997) Biochem. Biophys. Res. Commun.
232:379-382).
Thus, the methods of the invention can be used to generate modified VSV G
protein, thereby
2 5 generating new target cells for recombinant VSV.
There is also a need to generate tissue-specific adenoviruses. Since the
tropism of adenovirus is nonselective, tissue-specif c expression of
systemically administered
vectors can only be achieved by the use of a tissue-specific promoter/enhancer
that is small
enough to fit the insert capacity of the vector. Alternatively, tissue-
specific expression is
3 0 generated by ablating the native promiscuous tropism of adenovirus and
constructing new
tissue-specific domains using the methods of the invention. Generation of
tissue-specific
51

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
adenoviruses by recursive sequence recombination overcomes this non-selective
tropism
limitation of native adenovirus in the use of the vector in gene therapy.
Adenovirus binds to eukaryotic cells using a "fiber protein" which protrudes
from each of the twelve vertices of its icosahedral capsid. Serological and
mutagenesis
studies make it clear that the fiber, a homotrimer consisting of "staff' and
"knob" domains,
interacts with cellular receptors. The structure of the knob has been reported
by Xia (1994)
Structure 2:1259-1270. R. D. Gerard has used the structure of the
heterotrimeric knob to scan
this structure by SDM for mutations that reduce binding to the receptor
(personal
communication, 1996 CSH Gene Therapy meeting. These studies allow construction
of
mutants with abrogated or severely reduced ability to infect using the natural
receptor, which
is known to be expressed in many tissue types. This is a starting point from
which to evolve,
i.e., use the recursive sequence recombination methods of the invention, new
tissue
specificities for the adenovirus fibers which bind to cellular receptors. V.
Legrand (CSH
poster 184) and Dan von Seggery (CSH poster #223) have reported systems for
expressing
mutants of the fiber protein off of a small easily manipulated SV40 based
vector. These
constructs will support plaque formation by an adenovirus deleted for the
fiber gene. Legrand
used this system to fuse the 11 amino acid Gastrin Releasing Peptide (GRP) to
the C-terminus
of the fiber gene. LacZ+ adenoviral mutants expressing this fusion protein
were able to infect
cells expressing GRP receptor is a manner that was only 60% inhibitable by
soluble knob
2 0 protein (CSH poster 184), whereas viruses expressing the wild type protein
are about 90%
inhibitable. This was given as evidence that the interaction of GRP with its
receptor is
supporting infection of the host cells.
In one illustrative embodiment, to improve this adenovirus system using the
methods of the invention, a mutant fiber protein or a domain replacing the
knob that has lost
2 5 the ability to bind its native receptor is generated. Generation of
evolved fiber sequences by
recursive recombination generates a new adenovirus fiber or knob-associated
ligand with a
new specificity. Alternatively, libraries of mutant sequences can be inserted
onto the
C-terminus of the knob in a manner analogous to the GRP construct described
above.
Libraries of potential ligands can be randomly inserted throughout the "staff'
and/or "knob"
3 o domain. The entire knob can be randomly mutagenized and selected for
infection of desired
targets. Other exposed viral proteins, such as penton or hexon proteins, can
be modified with
libraries of insertion mutants. Libraries encoding short protein sequences can
be inserted in
52

CA 02268265 1999-03-24
WO 98/13485 PCT/US97117302
to adenovirus hexon protein and expressed on the surface of the adenovirus
virion as part of
the hexon (Crompton (1994) J. Gen. Virol. 75:133-139). Next, these modified
viruses
comprising the recursively evolved viral proteins are used to infect target
cells. Diversity and
modifications in viral protein affecting adenovirus tropism are selected for
by plaque
formation, or by cell sorting (FAGS), which can be based on transient
expression of a reporter
gene such as GFP.
Interaction of the fiber penton protein with an integrin on the target cell
surface, the alpha-v-integrin, provides a cell-virus stabilizing interaction
(it is known that one
cannot totally inhibit adenovirus infection with soluble fiber knob protein).
In the absence of
fiber penton-cell integrin interaction, there is a lower level of viral
infectivity. As a result of
this complexity in the mechnism which determines the cell specificity of
adenovirus, the
methods of the invention are used to coevolve multiple genes or domains on
adenovirus
which interact with their cognate receptors on target cells, such as the
penton fiber domain
which interacts with target cell alpha-v-integrin. Consequently, recursive
sequence
recombination of chosen viral genes, or of the whole virus, is a particularly
useful tool with
which to rapidly evolve tissue-specific adenovirus.
In another illustrative embodiment, the highly developed M13 technology is
used to evolve peptide ligands for receptors of interest on target cells.
Standard phage display
library technology is used to screen for peptide ligands capable of binding
purified receptor.
2 0 Alternatively, the libraries can be screened by panning against cells. The
affinity of these
ligands is rapidly evolved in M13. Pools of evolved ligands are then engrafted
onto target
sites on adenovirus, for example, C-terminal fusions to fiber protein. This
couples the power
of M13 selection to the adenovirus system, making it possible to make
libraries of the size
that could not be made with M 13 alone.
Screening is accomplished by contacting viruses containing recombinant
segments with a first population of cells for which infection by the virus is
desired and a
second population of cells, for which infection is not desired. Viral genomes
recovered from
the first population of cells are enriched for recombinant segments confernng
specificity for
that cell type. The first and second populations of cells can be present in
different tissues in
3 0 an organism. For example, one can infect a whole organism with the virus
and recover
recombinant segments from a subset of blood cells (this being the cell type
for which
infection is desired). Alternatively, one can infect a whole organism,
including humans,
53

CA 02268265 1999-03-24
WO 98/13485 PCT/ITS97/17302
suffering from a natural or induced cancer with virus and recover recombinant
segments from
the cancer cells. In a further variation, the first and second population of
cells are co-
cultivated with the virus in mixed cell culture. The two cell types, if they
are not readily
distinguishable by microscopic examination, can be distinguished by expression
of a marker,
such as green fluorescent protein or cell surface receptor in one cell type.
In the initial round
of screening, the existing host cells are usually present in excess (e.g., a
ratio of 90% existing
host cells to 10% desired target cells). The proportion of desired target
cells can be increased
in successive rounds.
The recombinant segments recovered from the population of cells for which
infection is desired are used as substrates in the next round of
recombination. Subsequent
rounds of screening are performed by the same principles.
In a variation of the above approach, a eukaryotic or bacterial virus is
modified
to have specificity for a given cell type by expressing a ligand as a fusion
protein with a viral
coat protein on the viruses outer surface. The ligand is chosen to have
affinity for a receptor
known to be present on the cell type of interest. For example, the EGF family
of proteins
encompasses several polypeptides such as epidermal growth factor (EGF),
transforming
growth factor alpha (TGF alpha), amphiregulin (AR) and heregulin (HRG-beta 1
), which
regulate proliferation in breast cancer cells through interaction with
membrane receptors.
Han (1995) Proc. Natl. Acad. Sci. USA 92:9747-9751, reported that Moloney
murine
2 0 leukemia virus can be modified to express human heregulin fused to gp70,
and the
recombinant virus infects certain human breast cancer cells expressing human
epidermal
growth factor receptor.
This principle can be extended to other pairs of virus expressing a ligand
fusion protein and target cell expressing a receptor. For example, filamentous
phage can be
engineered to display antibody fragments (e.g., Fab or Fv) having specifc
binding affinity for
virtually any chosen cellular receptor. Binding specificity of ligand to
receptor can be
optimized by recursive recombination of the segment of the viral genome
encoding the
ligand, and screening using first and second populations of cells as discussed
above.
Although viral vectors are most amenable to evolution/recursive
3 0 recombination to acquire new or altered tissue specificity, the same
principles can be applied
to nonviral vectors. Such vectors can be engineered to contain specific uptake
sequences
thought to favor uptake by specific target cells. Alternatively, variants of
nonviral vectors can
54

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
be recombined without prior knowledge of sequences that might mediate uptake.
For
example, the starting substrates can be random sequences. Recombination
products are
contacted with first and second populations of cells as described above under
similar
conditions to those contemplated for use of the vector. For example, if a
vector is to be used
packaged in liposomes, screening is performed with vectors containing
recombinant segments
packaged as Iiposomes. Again, vectors containing recombinant segments are
recovered from
the population of target cells and these segments are used in the next round
of recombination.
(I7 Improved Uptake of DNA Mediated by Evolved DNA Binding Proteins
The efficiency and specificity of uptake of vector nucleic acid uptake by a
given cell type can be improved by coating the vector with an
evolved/recursively
recombined and modified protein that binds to the nucleic acid. The vector can
be contacted
with the modified protein in vitro or in vivo. In the latter situation, the
protein is expressed in
cells containing the vector, optionally from a coding sequence within the
vector. The nucleic
acid binding proteins to be evolved usually have nucleic acid binding activity
but do not
necessarily have any known capacity to enhance or alter nucleic acid DNA
uptake.
In this embodiment, DNA binding proteins that are modified by the methods
of the invention include transcriptional regulators, enzymes involved in DNA
replication
(e.g., recA) and recombination, and proteins that serve structural functions
on DNA (e.g.,
histones, protamines). Other DNA binding proteins can include the phage 434
repressor, the
2 0 lambda phage cI and cro repressors, the E. coli CAP protein, myc, proteins
with leucine
zippers and DNA binding basic domains such as fos and jun; proteins with 'POU'
domains
such as the Drosophila paired protein; proteins with domains whose structures
depend on
metal ion chelation such as Cys2I-Iis~ zinc fingers found in TFIIIA, Zn2(Cys)6
clusters such as
those found in yeast Gal4, the Cys3His box found in retroviral nucleocapsid
proteins, and the
2 5 Zn2(Cys)g clusters found in nuclear hormone receptor-type proteins; the
phage P22 Arc and
Mnt repressors (see Knight (1989) J. Biol. Chem. 264:3639-3642; Bowie (1989)
J. Biol.
Chem. 264:7596-7602). RNA binding proteins are reviewed by Burd (1994) Science
265:615-621, and include HIV Tat and Rev.
As in other embodiment of the invention, evolution of DNA binding proteins
3 0 toward acquisition of improved or altered uptake efficiency is effected by
recursive cycles of
recombination and screening. The starting substrates can be nucleic acid
segments encoding
natural or induced variants of one or nucleic acid binding proteins, such as
those mentioned

CA 02268265 1999-03-24
WO 98/13485 PCT1US97/17302
above. The nucleic acid segments can be present in vectors or in isolated form
for the
recombination step. Recombination can proceed through any of the formats
described in
Section II.
For screening purposes, the recombined nucleic acid segments should be
inserted into a vector, if not already present in such a vector during the
recombination step.
The vector encodes a selective marker capable of being expressed in the cell
type for which
uptake is desired. If the DNA binding protein being evolved recognizes a
specific binding
site (e.g., lacI binding protein recognizes IacO}, this binding site can be
included in the
vector. Optionally, the vector can contain multiple binding sites in tandem.
The vectors containing different recombinant segments are transformed into
host cells, usually E. coli, to allow recombinant proteins to be expressed and
bind to the
vector encoding their genetic material. Most cells take up only a single
vector and so
transformation results in a population of cells, most of which contain a
single species of
vector. After an appropriate period to allow for expression and binding, cells
are lysed under
mild conditions that do not disrupt binding of vectors to DNA binding
proteins. For example,
a lysis buffer of 35 mM HEPES {pH 7.5 with KOH}, 0.1 mM EDTA, 100 mM Na
glutamate,
5% glycerol, 0.3 mg/mi BSA, 1 mM DTT, and 0.1 mM pMSF) plus lysozyme (0.3 mi
at 10
mg/ml) is suitable (see Schatz et al., US 5,338,665). The complexes of vector
and nucleic
acid binding protein are then contacted with cells of the type for which
improved or altered
2 o uptake is desired under conditions favoring uptake (e.g., for eukaryotic
cells, recipient cells
can be treated with calcium phosphate or subjected to electroporation).
Suitable recipient
cells include the human cell types that are common targets in gene therapy,
discussed
elsewhere in this application.
After incubation, cells are plated with selection for expression of the
selective
2 5 marker present in the vector containing the recombinant segments. Cells
expressing the
marker are recovered. These cells are enriched for recombinant segments
encoding nucleic
acid binding proteins that enhance uptake of vectors encoding the respective
recombinant
segments. The recombinant segments from cells expressing the marker can then
be subjected
to a further round of selection. Usually, the recombinant segments are first
recovered from
3 0 cells, e.g., by PCR amplification. The recombinant segments can then be
recombined with
each other or with other sources of DNA binding protein variants to generate
further
56

CA 02268265 1999-03-24
WO 98/i3485 PCTNS97/17302
recombinant segments. The further recombinant segments are screened in the
same manner
as before.
In a variation of the above procedure, a binding site recognized by a DNA
binding protein can be evolved instead of, or as well as, the DNA binding
protein. DNA
binding sites are evolved by an analogous procedure to DNA binding proteins
except that the
starting substrates contain variant binding sites and recombinant forms of
these sites are
screened as a component of a vector that also encodes a DNA binding protein.
Evolved nucleic acid segments encoding DNA binding proteins and/or
evolved DNA binding sites can be included in gene therapy vectors. If the
affinity of the
DNA binding protein is specific to a known DNA binding site, it is sufficient
to include that
binding site and the sequence encoding the DNA binding protein in the gene
therapy vector
together with such other coding and regulatory sequences are required to
effect gene therapy.
In some instances, the evolved DNA binding protein may not have a high degree
of sequence
specificity and it may be unknown precisely which sites on the vector used in
screening are
bound by the protein. In these circumstances, the gene therapy vector should
include all or
most of the screening vector sequences together with additional sequences
required to effect
genetherapy.
An exemplary selection scheme is shown in Figure 2. The lower left portion
of the Figure shows two vectors, each having the same marker and DNA binding
site, the
2 0 vectors differing in the recombinant segment encoding a DNA binding
protein. The vectors
are transfected into E. toll cells. The vectors are expressed in the cells to
produce DNA
binding proteins, which differ between the different cells. The recombinant
binding proteins
complex with the vectors encoding them and these complexes are preserved after
cell lysis.
The complexes are then contacted with a recipient eukaryotic cell. The
eukaryotic cell bears
2 5 several different cell surface receptors, one of which can interact with
one of the DNA
binding proteins to facilitate uptake of DNA. Selection for expression of the
selection marker
on the vector identifies cells transformed with vector. These cells are
enriched for
recombinant segments conferring enhanced DNA uptake.
Improved Intracellular Stability of a Vector
3 o Vectors with greater and improved cell retention, intracellular stability
and
expression properties can be developed using the recursive recombination
methods of the
invention. In many gene therapy methods, it is desirable that the vector be
stably maintained
57

CA 02268265 1999-03-24
WO 98/13485 PCT/US97l17302
in target cells and thereby be capable of indefinite expression. This is the
case for both viral
and nonviral vectors. Substrates and recombination formats for evolution of
vectors toward
improved retention can be chosen according to the principles described above.
If the
substrates are fragments of vector genomes, the recombination products are
reinserted into
vector genomes before screening. The vector genomes can often contain a
selective marker
replacing or fused to the therapeutic coding sequence carried by the vector in
actual use. For
screening, vector genomes containing recombinant segments are introduced into
cells, if they
are not already so present as a result of in vivo recombination. The cells are
grown for a
number of generations without selection for the marker, thereby reflecting the
situation in
l0 vivo, in which it is typically not possible to select for retention of a
therapeutic gene. After an
appropriate period of growth, selection for the marker is applied and
surviving cells
recovered. These cells can contain vectors harboring recombinant segments
conferring the
property of improved retention (i, e., recombinant segments stably maintained)
in a cell. In
some instances, the properties of improved retention, at least in part, a
consequence of
improved, more stable integration into the cellular genome. Recombinant
segments having
the property of improved replication, retention and/or stability are recovered
from cells, and
subjected to a further round of recombination, either with each other and/or
with fresh
substrates to generate further recombinant segments. These are screened in the
same manner
as the previous recombinant segments.
2 0 1Kl Reduced Immuno~genicity of Vectors
Protein and nucleic acid sequences with reduced immunogenicity can be
developed using the recursive recombination methods of the invention.
Immunogenicity is a
particular concern with viral vectors, since a host immune response, including
CTL mediated
and humoral responses, can prevent a virus from reaching its intended target
particularly in
repeated administrations. Cellular immune responses preventing a virus from
reaching its
intended target can also be induced against nonviral vectors administered in
naked form or
shielded with a coat such as liposomes.
Host immune responses which eliminate infected cells is also a major problem
in gene therapy. CTLs are primarily responsible for the elimination of
infected cells,
3 o although the problem can also be partly or entirely antibody-mediated. The
recursive
recombination methods of the invention can be used to modify a virus to reduce
this
(primarily cellular) immunity against virally infected cells. In a variation
of this embodiment,
58

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
for adenovirus-mediated gene transfer, adenovirus late gene expression is
reduced by
mutations induced by the methods of the invention to reduce CTL responses
which contribute
to the elimination of virus-infected cells. Thus, the problem of transient
retention of virus
which can be seen in adenovirus-mediated gene transfer is alleviated.
Substrates and formats for recombination generally follow the principles
discussed above. In general, regions of the viral genome encoding outer
surface proteins
provide the most likely initial substrates for evolution toward reduced
immunogenicity.
Alternatively, the whole vector genome can be included as an initial substrate
for
recombination. Recombinant viral genomes should be packaged as viruses before
screening,
and nonviral genomes should be prepared in the proposed composition for
therapeutic
administration (e.g., liposomes).
Viruses containing recombinant genomes or nonviral genomes appropriately
formulated are administered to a mammal, such as a mouse, rat, rabbit, pig,
horse, primate or
human, and surviving viruses or nonviral genomes are recovered after an
appropriate interval.
Often the administration is i.v. and surviving viruses and nonviral genomes
are recovered
from the blood. Surviving viruses and nonviral genomes are enriched for
recombinant
segments conferring the property of reduced immunogenicity. These recombinant
segments
are used as some or all of the substrates in the next round of recombination.
Subsequent
rounds of selection follow the same format.
2 0 In a variation of the above format, antibodies are collected from mammals
immunized with the viral library, and immobilized on a column. Another aliquot
of the viral
library, or a derivative library resulting from a further round of
recombination, can then be
applied to the column and viruses passing through the column collected. These
viruses are
enriched for viruses with low immunogenicity.
2 5 In a variation of this method for nonviral vectors, the therapeutic
expression
product of the vector is expressed as a fusion protein joined to a DNA binding
pratein that
has affinity for a sequence on the vector. In this way, at least some of the
expression product
is maintained in physical proximity with the vector producing it. Thus, immune
responses
directed against the expression product also remove the vector sequence.
Accordingly,
3 0 recovery of vector sequences surviving a period of time in an animal,
enriches both vector
sequences that themselves have low immunogenicity and which encode expression
products
with low immunogenicity.
59

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
l~,l R~,duced Toxicity of Vectors
Protein and nucleic acid sequences with reduced cellular toxicity can be
developed using ther recursive recombination methods of the invention.
Toxicity caused by
viral gene expression is sometimes a concern when using viral vectors in gene
therapy. The
methods of the invention can be used to induced and select for multiple
combinations of
mutations blocking viral DNA replication and gene expression in vivo. To
produced the
crippled viruses in vitro, these mutations should be conditional mutations,
such as
temperature sensitive or nonsense mutations so that the mutant viruses can be
propagated in
vitro under permissive conditions. The multiplicity and hence redundancy of
the conditional
mutations prevents the mutant virus from reverting back to the wildtype
genotype or
phenotype.
(Ml Improved Specificity of Inte ration
Vector sequences with improved specificity of integration can be developed
using the recursive recombination methods of the invention. For example, AAV
is known to
integrate preferentially at a site in chromosome 19q13.3. Integration at this
site is
advantageous since the presence of an exogenous DNA sequence at this site does
not appear
to have any adverse effect on expression of endogenous cellular genes. It is
therefore
desirable to be able to increase the specificity of AAV to this site.
The starting substrates for recombination are AAV vectors including at least
2 0 ITRs and, optionally, a rep gene, since the latter may have a role in site-
specific
recombination. Genes from other viruses known or believed to have a role in
site specific
integration can also be included. Preferably the genomes include a marker
sequence.
Recombination proceeds through any of the recombination formats previously
discussed to
produce a library of AAV viruses having different recombinant segments in
their genomes.
The AAV viruses are used to infect appropriate target cells. Cells having
taken up AAV
DNA can be recognized from expression of the marker. Genomic DNA is isolated
from these
cells, and a region centered on the intended site of integration is amplified
by PCR. The
amplified regions are enriched for recombinant segments conferring the desired
property of
site-specific integration. These recombinant segments form the starting
materials for the next
3 0 round of recombination.
Analogous principles apply to other viral vectors and, indeed, nonviral
sequences and vectors. For example, as discussed above, one embodiment of the
invention

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
utilizes site-specific integration systems to target a recombinant sequence of
interest to a
specific, constant location in the genome. A preferred embodiment uses the
Cre/LoxP or the
related FLP/FRT site-specific integration system. The Cre/LoxP system uses a
Cre
recombinase enzyme to mediate site-specific insertion and excision of viral or
phage vectors
into a specific palindromic 34 base pair sequence ("LoxP site"). The recursive
sequence
recombination methods of the invention can be used to modify these systems,
such as to
improve specificity of integration, create alternate, specific sites of
integration, modify
recombinase activity, and the like.
In a further embodiment, it is not necessary that the starting vector have any
preferred integration site. If this is the case, a suitable chromosomal site
unlikely to interfere
with expression of other genes is chosen, and successive cycles of
recombination and
selection performed until a vector has evolved to integrate preferentially at
that site.
(N~ Improved Resistance to Microor ang isms
The recursive recombination methods of the invention can also be used to
develop new or improve upon known inhibitors of microbial and viral infection,
including
trans-dominant inhibitors of microbial and viral replication and gene
expression. In some
gene therapy applications, the vector can encode a product that is an
inhibitor to a
microorganism, such as a virus. Because of the complexity of viral life cycles
and the
intrinsic mutability of viruses, recursive sequence recombination is a
practical tool for
2 0 evolving protective antiviral constructs with improved potency and/or new
or improved
specificities. This can be accomplished using any variety of mechanisms. For
example, the
gene therapy vector can encode an antisense RNA that blocks expression of a
viral or other
pathogen's mRNA. The antisense RNA can be designed to bind to a key regulatory
sequence,
such as a promoter, or to the coding sequence, or both. Alternatively, the
vector can encode a
2 5 protein that is inhibitory to the replication or gene expression of a
pathogen. For example, a
number of gene therapy strategies have been designed with the intent of
inhibiting HIV-1
replication in mature T cells. As T cells are products of hematolymphoid
differentiation,
insertion of antiviral genes into hematopoietic stem cells serves as a vehicle
to confer
long-term protection in progeny T cells derived from transduced stem cells.
One such
3 0 "cellular immunization" strategy utilizes the gene coding for the HIV-1
rev trans-dominant
mutant protein RevMlO which has been demonstrated to inhibit HIV-1 replication
in T-cell
lines and in primary T cells; as described in Bonyhadi (1997) J. Virol.
71:4707-4716; Nabel
61

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
(1996) Gene Therapy, abstract 361, CSH. HIV-I tat and rev mutants have also
been
suggested as potential intracellular, traps-dominant inhibitors of HIV-1
replication, Caputo
(1997) Gene Ther. 4:288-295. Another candidate for development by the methods
of the
invention is the traps-acting transcriptional regulatory protein I kappa B
alpha, which can act
as a cellular inhibitor of human retroviral replication through a mechanism
independent of its
effect on HIV transcription, see Wu (1995) Proc. Natl. Acad. Sci. USA 92:1480-
1484.
Repeats of inhibitors derived from viral fragments, such as poly-TAR
constructs, can also be
used as inhibitors of HIV-1 gene expression. TAR is an RNA stem-loop structure
bound by
activators or inhibitors of HIV-1 gene expression. TAR can be used to mediate
(for example,
l0 saturate) cellular factor/RNA interactions, and it has been suggested that
transcriptional
activators (such as Tat) action might be inhibited by such competing TAR
reactions in vivo;
see Baker (1994) Nucleic Acids Res. 22:3365-3372. The recursive recombination
methods of
the invention can develop and improve upon these and related intracellular
inhibitory systems
There are also many examples where a protein from one virus or viral product
can be inhibitory to the development of another. Woffendin ( 1994) Proc. Natl.
Acad Sci.
USA 91:11581-I 1585. In particular, at least one protein from adeno-associated
virus (AAV)
is known to be inhibitory to HIV. The large rep gene products, Rep78 and
Rep68, of AAV
are pleiotropic effector proteins which are required for AAV DNA replication
and the
traps-regulation of AAV gene expression. Apart from these essential functions,
these rep
2 o products are able to inhibit the replication and gene expression of HIV-1
and a number of
DNA viruses. Batchu ( 1995 ) FEBS Lett. 367:267-271; Antoni ( 1991 ) J. Virol.
65:396-404.
The recursive recombination methods of the invention can develop new and
improve upon
these inter-viral inhibitory proteins.
The present invention provides a means for improving the inhibitory qualities
of the anti-sense RNAs and proteins described above and also for identifying
new inhibitory
agents. The improvement to known inhibitory reagents can reside in several
aspects, such as
improved expression, improved stability or altered fine-binding specificity.
It is not
necessary in the present methods to know which of these contributory
properties is being
improved; rather the selection is for the ultimately desired property of
microorganism
3 0 resistance.
For evolution of known inhibitory agents, substrates for recombination and
recombination formats are selected according to the principles discussed
above. The
62

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
substrates can be viral vector genomes or the parts thereof encoding the
inhibitory agents and
associated regulatory sequences. Initial diversity in recombination substrates
can be natural
or induced. After a round of recombination, the recombinant segments are
introduced into
cells (if they are not already in cells as a result of in vivo recombination)
and the cell are
contacted with the microorganism for which protection is desired. Cells
surviving exposure
to the microorganism are enriched for recombinant segments conferring
resistance to the
microorganism. These recombinant segments form some or all of the substrates
for the next
round of recombination.
Similar principles can be applied for de novo identification of inhibitory
agents
to be expressed from gene therapy vectors. More rounds of recombination and
screening can
be required to obtain satisfactory results. For example, sequences coding for
viral proteins
from the virus to be inhibited or other viruses provide suitable initial
substrates for
recombination. The coding sequences can be obtained from the same or different
viruses and
natural diversity can be augmented by inducing additional mutations, e.g., by
error-prone
PCR, as described above. Recombination and screening are also performed as
described
above.
In an illustrative embodiment, a library of mutants is constructed based on
candidate construct(s), examples of which are described above. The libraries
are transduced
or transfected into target cells. The cells are challenged with the
microorganism of interest.
2 0 Resistant cells are isolated based on, for example, survival against
cytopathic virus or lack of
expression of viral encoded genes, which can include inserted marker genes
such as GFP.
These methods are used to detect cells in which viral replication or gene
expression has been
blocked. FACS or panning with an antibody against a virally encoded or induced
surface
epitope is used in a positive selective step. Genes encoding resistance factor
are recovered,
for example, by PCR. The recovered genes can be subjected to further rounds of
recursive
sequence recombination, as described above, until a desired level of
protection against the
microorganism is achieved
Further illustrative examples of anti-viral mechanisms which can be improved
by the methods of the invention include anti-viral ribozyme systems. For
example, one or
3 0 more ribozymes can be targeted against a viral RNA. Adenoviruses have been
used to deliver
anti-hepatitis C ribozymes; see Lieber ( 1996) J. Virol. 70:8782-8791; Ohkawa
( 1997) J.
Hepatol. 27:78-84. HIV-1 Rev response element (RRE) region-specific hammerhead
63

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
ribozymes will completely inhibit HIV-1 replication, see Duan (1997) Gene Ther
4:533-543.
Sendai virus polycistronic P/C mRNA can also be cleaved by ribozymes; Gavin (
I 997) J.
Biol. Chem. 272:1461-1472.
Anti-viral cytokines can also be improved by the methods of the invention.
For example, wild type or chimeras of wild type interferons such as the IFN
alpha 17, IFN
beta and IFN gamma constructs can be subjected to recursive sequence
recombination. These
sequences can placed be under the control of a virus-activated promoter, such
as an HIV
mini-LTR; see Mehtali (1996) Gene Therapy, abstract #364, CSH. For example,
cell lines
stably carrying IFN transgenes under the positive control of the HIV-1 Tat
protein are highly
resistant to HIV-1 replication in vitro. This antiviral resistance is
associated with a strong
induction of IFN synthesis immediately following the viral infection. However,
IFN-gamma-transfected cells permitted HIV-1 infection in vivo despite the
induction of a
high level of IFN-gamma secretion, see Sanhadji (1997) AIDS I 1:977-986. The
methods of
the invention can be used to develop this anti-viral system for potency and
effectiveness in
vivo.
The methods of the invention can be used to develop single chain or Fab
antibody fragments directed intracellularly to viral components; Marasco (
1996) Gene
Therapy, abstract 160, CSH. For example, one strategy for somatic gene therapy
to treat
HIV-I infection is by intracellular expression of an anti-HIV-I Rev single
chain variable
fragment (Sfv); Duan (1997) Gene Ther, supra. Intracellular expression of Sfvs
which bind
to HIV integrase catalytic and carboxy-terminal domains results in resistance
to productive
HIV-I infection. This inhibition of HIV-1 replication is observed with Sfvs
localized in either
the cytoplasmic or nuclear compartment of the cell. See Levy-Mintz (1996) J.
Virol.
70:8821-8832. The expression of anti-reverse transcriptase (RT) Sfv in T-
lymphocytic cells
specifically neutralizes the RT activity in the preintegration stage and
affects the reverse
transcription process, an early event of the HIV-I life cycle. Blocking the
virus at
these early stages dramatically decreased HIV-1 propagation, as well as the
HIV-I-induced
cytopathic effects in susceptible human T lymphocytes, by impeding the
formation of the
proviral DNA. See Shaheen ( 1996) J. Virol. 70:3392-3400. The methods of the
invention
3 o can further develop the potency and range of such anti-viral,
intracellular antibody fragments.
Improved virus-binding aptamers or peptide ligands directed to viral
components, as those described above, can also be further developed by the
methods of the
64

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
invention. For example, RNA aptamers that recognize a peptide fragment of
human HIV-1
Rev were found to bind the free peptide more tightly than a natural RNA
ligand, the
Rev-binding element, see Xu (1996) Proc. Natl. Acad. Sci. USA 93:7475-7480;
Symensma
(1996) J. Virol. 70:179-187. Aptamer sequences isolated from single-stranded
DNA
preparations have thrombin inhibitory activity, indicating that thrombin-
inhibitory aptamers
are present in the mammalian genome and may constitute an endogenous
antithrombin
system. Analogously, the recursive sequence methods of the invention can be
used to further
identify, develop and improve aptamer sequences useful as anti-microbial
agents, or for gene
therapy in general.
~Q) Viral Packaging Cell Lines
The recursive sequence recombination methods of the invention can also be
used to develop new and improved viral packaging cell lines Viral vectors used
in gene
therapy are usually packaged into viral particles by a packaging cell line.
The vectors
typically contain the minimal viral sequences required for packaging and
subsequent
integration into a host, other viral sequences being replaced by an expression
cassette for the
protein to be expressed. The missing viral functions are supplied in trans by
the packaging
cell line. For example, AAV vectors used in gene therapy typically only
possess ITR
sequences from the AAV genome which are required for packaging and integration
into the
host genome. Viral DNA is packaged in a cell line, which contains a helper
plasmid
2 0 encoding the other AAV genes, namely rep and cap, but lacking ITR
sequences. The cell line
is also infected with adenovirus as a helper. The helper virus promotes
replication of the
AAV vector and expression of AAV genes from the helper plasmid. The helper
plasmid is
not packaged in significant amounts due to a lack of ITR sequences.
Contamination with
adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more
sensitive than
2 5 AAV. AAV recombinants are generally produced by transient co-transfection
methods since
it has proven difficult to generate stable packaging cell lines (Maxwell
(1997) J. Virol.
Methods 63:129-136).
The goals in improving packaging cell lines include generating stable
packaging cell lines; increasing the yield of AAV vector packaged; decreasing
the ratio of
3 0 AAV progeny to helper virus; and reducing the toxicity of the rep gene to
the packaging cell,
which in turn leads to a greater yield of AAV. The leading candidate genes for
evolution/
modification by the methods of the invention are the AAV replication (rep) and
capsid (cap)

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
genes, which can be present on the AAV helper plasmid. Overexpression of the
rep gene can
decrease AAV DNA replication and severely inhibit cap gene expression and
reduced
rep level enhances cap gene expression and supports normal rAAV DNA
replication. Thus,
recursive recombination modification of rep genes and their expression can
generate
increased AAV vector production, see Li (1997) J. Virol. 71:5236-5243.
These and related sequences can be subject to recursive sequence
recombination according to the general principles discussed. That is, variant
forms of these
genes are recombined, either in vivo or in vitro, and cells containing
recombinant segments
resulting from recombination are screened for a desired property, such as
stable packaging
cell lines; yield of packaged AAV; increased viability of cells; or, low yield
of helper virus
relative to packaged AAV. The same principles can be applied to evolve genes
in the helper
adenovirus, either concurrently or consecutively with the evolution of AAV
genes on the
helper plasmid.
Cellular genes in the packaging cell line affecting packaging can also be
1 S evolved even without knowing what these genes are. This is achieved by
transforming the
packaging cell line with a library of genes, some of which will undergo
recombination with
cognate genes in the packaging cell line. The library of genes can be obtained
from another
type or species of cell or can be a mixture of several types and species
and/or can have
diversity induced by processes such as error-prone PCR. Cells containing
recombinant genes
2 0 are screened for improved packaging properties, such as increased yield of
AAV virus.
Optionally, a further library can be transformed into the cells surviving
screening in a
previous round. Alternatively, the pool of surviving cells can be divided in
two, and DNA
isolated from one half and used to transform the other half. In this way, the
best recombinant
segments identified in the first round of screening undergo recombination with
each other in
2 5 the second round of recombination.
EXAMPLES
Example 1 ~ M13 scFv Library
This example shows in vivo panning of libraries of bacteriophage displaying
3 0 scFv for localization to a predetermined cell type, such as a xenogeneic
neoplasm. A scFv
antibody-phage display library was constructed as described in Crameri (1996)
Nature
Medicine 2:100-102. After growth of the phage library on E coli TGI in LB
containing 50
66

CA 02268265 1999-03-24
WO 98/13485 PCT/US97117302
~g/ml kanamycin, bacterial cells were removed by centrifugation and the phage
precipitated
by addition of PEG to 4% and NaCI to 0.5 M final concentration. After one hour
incubation
on ice, the solution was centrifuged at 8,000 x g for 30 minutes, and the
pellet resuspended in
Dulbecco's phosphate-buffered saline (DPBS).
Male Sprague-Dawley rats were anesthetized and phage were injected
intravenously and blood sampled arterially via ipsilateral femoral arterial
catheters. EDTA
was used in blood samples to reduce coagulation. Blood samples were taken
immediately
before administration of phage and at 5, 30, 60, 120, and 240 minutes post-
injection of 7.6 x
10" colony forming units. Phage titers were determined by dilution of whole
blood in DPBS
and infection of E. coli TG1 to assay colony forming units of M13. Four
repetitions of the
protocol were performed. It was found that M 13 bacteriophage remained stable
and
infectious (to E. toll) with a half life of six hours in rat blood after in
vivo injection.
Example 2: Panning of M 13 scFv Library for Specific Localization
A scFv antibody-phage display library is administered to mice having
transplantable human tumor grafts. After a suitable incubation time, tumor
tissue is harvested
and phage are eluted from the harvested tissue by homogenization of the tissue
sample.
An aliquot of the recovered phage is subjected to at least one additional
cycle
of administration and selection in vivo by the same protocol.
2 0 An aliquot of the recovered phage is used to purify DNA and the recovered
DNA is recursively recombined by shuffling in vitro, and the resultant
population of shuffled
M13 genomes is introduced into E. toll and packaged; a library of shuffled M13
species is
recovered and administered to mice for at least one additional cycle of
administration and
selection in vivo by the same protocol.
2 5 An aliquot of the recovered phage is used to infect E. toll at a high
multiplicity
of infection to recursively recombine M13 genomes in vivo by shuffling, and
the resultant
population of shuffled M13 genomes is introduced into E. toll and packaged; a
library of
shuffled M13 species is recovered and administered to mice for at least one
additional cycle
of administration and selection in vivo by the same protocol.
67

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
Example 3: Evolution of the MGMT gene
This example illustrates evolution of the MGMT gene to confer improved
properties for protection of human bone marrow against alkylating agents. The
wild-type
human MGMT cDNA on a high copy number plasmid was amplified by PCR and
randomly
fragmented with DNase. Small fragments (50-100bp) were reassembled into full-
length
fragments by Taq DNA polymerase without outside primers in a process that
induces point
mutations in a rate proportional to the size of the starting fragments, see
Stemmer ( 1994)
Proc. Natl. Acad. Sci. USA 91:10747-10751. Shuffling the entire gene, which
encodes 207
amino acids, allows mutagenesis of all regions of the protein including the
functionally
important DNA-binding region (Kanugula (1995) Biochemistry 34:7113-7119). Full-
length
fragments were cloned back into the vector and transformed into
alkyltransferase-deficient E.
toll (strain GWR111, ada ogt) (Rebeck (1991) J. Bacteriol. 173:2068-2076).
Relatively
large numbers of mutations were created to increase diversity and because
inactive variants
can be eliminated with stringent genetic selection by alkylating agents. This
selection
involves treating the bacteria with the methylating agent MNNG three
sequential times, each
separated by a one-hour recovery period during which the bacteria are allowed
to make more
MGMT. The triple selection kills cells having inactive MGMT and preferentially
selects for
proteins having improved expression and/or activity of MGMT.
An improved human MGMT gene was also generated using both natural and
2 0 unnatural -encoding sequence diversity. Unnatural diversity was created by
the random
fragmentation of the human MGMT (wild-type MGMT cDNA was generously provided
by
Dr. S. Mitra, University of Texas, Galveston; see Tano ( 1990) "Isolation and
structural
characterization of a cDNA clone encoding the human DNA repair protein for
06-alkylguanine," Proc. Natl. Acad. Sci. USA 87:686-690, for cDNA and protein
sequences
and for residue numbering). This was followed by the reassembly of fragments
in a
mutagenic DNA shuffling reaction. Active variants, selected for their ability
to confer
MNNG resistance to alkyltransferase-deficient E. toll, were pooled, remutated,
and
recombined in subsequent cycles of shuffling (the alkyltransferase-deficient
(ada ogt) E toll
strain GWR111 was provided by L. Samson, Harvard University, Cambridge, MA;
Rebeck
3 0 ( 1991 ) J. Bacteriol. 173 :2068-2076). Two cycles of conventional DNA
shuffling were used
to build up the unnatural diversity.
68

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
The wild-type human alkyltransferase (MGMT) cDNA was subcloned into
pUC 118 plasmid (New England Biolabs, Beverly, MA) and a translationally
silent XhoI site
created at coding nucleotide residue number 380 (Taro (1990) supra, for
residue numbering).
The flanking non-coding sequences were removed from that construct and an E.
coli
ribosome-binding site added via PCR amplification with oligos 1 and 2 (see
below) and
inserted into the EcoRI-HinDIII sites of pUCI 18.
Oligo # I : 5'-GCATCCGAATTCCTTAAGGAGGGGAAAAATGGACAAGGATTG-3'
Oligo #2: 5'-CCGCTAAAGCTTCATACTCAGTTTCGGCCAG -3'
This construct is designated "pFC 14." The sequence of the entire MGMT gene in
pFC 14 was
verified, as was its ability to complement GWR1 I 1. A non-functional dummy
vector was
constructed by replacing the active site-encoding region between the XhoI and
PinAI sites
(nucleotide residue numbers 380 to 521 (Tano (1990) supra, for residue
numbering) with a
synthetic stuffer duplex made by annealing oligos 3 and 4 (below).
Oligo #3: 5'-TCGAGCCCCAGGCCTCCGCA-3'
Oligo #4: 5'-CCGGTGCGGAGGCCTGGGGC-3'
The inactivity of this gene was verified by its inability to complement
GWR111. The dummy
vector, with the shortened MGMT removed, was used as a cloning vector for
library
construction to reduce the possibility of contamination by wild-type MGMT.
The general procedure for creating randomized gene libraries by random
2 o fragmentation and reassembly was used as described in Stemmer ( 1994)
Proc. Natl. Acad.
Sci. USA 91:10747-10751; and Stemmer (1994) Nature 370:389-391. The starting
material
was a 1.2 kbp PCR product made from pFCl4, generated using the outside primers
oligo #5:
5'-AAGAGCGCCCAATACGCAAA-3', and oligo #6: 5'-
TAGCGGTCACGCTGCGCGTAA-3', and Taq DNA polymerase (Promega). This product
contained the human MGMT plus pFCl4 flanking sequence from which 50-300 by
random
fragments were prepared and reassembled with Tag DNA polymerase, as in Stemmer
( 1994)
Proc. Natl. Acad .Sci. USA, supra, and Stemmer (1994) Nature, supra.
Reamplification with
the nested primers oligo #7: 5'-ATGCAGCTGGCACGACAGGTTT-3' and oligo #8: 5'-
TACAGGGCGCGTACTATGGTT-3', gave a 980-by fragment which was treated with EcoRI
3 0 and HinDIII. The resulting 650-by fragment was ligated into the dummy
vector described
above. The ligation mixture was electroporated into GWR11 l, yielding
libraries of 105 per
69

CA 02268265 1999-03-24
WO 98/13485 PCT/ITS97/17302
cycle from which active clones were selected. Selection was done as described
in Christians
( i 996) Proc. Natl. Acad .Sci. USA 93:6124-6128, with the exception of
omission of the
inducer isopropyl-beta-thiogalactopyranoside. Bacteria in culture were treated
with 3
sequential doses of MNNG, each separated by a 1-hour recovery period. After
the third dose
all cells were spread on plates. The next day colonies were pooled, and the
MGMT DNA for
the next cycle was prepared by PCR with oligos #5 and #6 (above). This
procedure was
repeated for a total of 6 cycles. The MNNG treatment was made progressively
more stringent
as the shuffling progressed, starting at 3 x 10 ug/ml MNNG up to as much as 50
ug/ml in later
cycles. Likewise, fewer colonies were picked for shuffling in later cycles.
The natural diversity of four known mammalian alkyltransferases - rat, mouse,
hamster, and rabbit - was atso used to generate sequence diversity in the
improved human
MGMT gene. An alignment of their protein sequences, as shown in Figure 4.
reveals regions
of extensive homology as well as regions of diversity. There exist 2 x l Ozg
combinations of
known natural amino acid substitutions from mammalian alkyltransferases (52
positions with
2 amino acids represented, 24 positions with 3 amino acids, and 2 positions
with 4 amino
acids = 252 x 3z4 x 42). This diversity was exploited through the use of 21
degenerate
oligonucleotides (Figure 3). These oligos were mixed together in equal
proportions to create
one diverse pool, which was mixed with the DNA fragments during the reassembly
reactions
in the third and fourth cycles. Several different molar ratios of
oligos:fragments were made,
and it was observed that high concentrations of oiigonucleotides inhibited
reassembly,
probably because the large number of base pair mismatches overwhelmed the
polymerase. Of
those mixtures giving proper reassembly, as judged by correct product size
after
reamplification, the one containing the highest proportion of oligos, a molar
ratio of 1 oligo:4
fragments, was chosen for further cycling. Annealing of each oligonucleotide
to the human-
derived MGMT sequence was enabled by 20 nucleotides of homology on both sides
flanking
the degenerate or non-human sequence. Control PCRs demonstrated that all
oligonucleotides
were approximately equally capable of hybridizing to the human sequence.
In the third round of shuffling, the oligonucleotides were combined with the
sequences generated by oligonucleotides having "unnatural diversity," that is,
the pooled
3 0 human MGMT clones that survived cycle 2. Conditions were varied in an
attempt to
incorporate the oligonucleotides and maximize diversity while maintaining the
correct size of

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
the assembled product. The largest molar ratio of oligonucleotide:fragment to
allow correct
assembly was 1:4. Because of the limitation in the ratio, the "oligo spiking"
was repeated in
cycle 4. The pools in cycles 3 and 4 were thus hybrids containing randomly
mutated human-
derived sequence as well as different combinations of mammalian MGMT gene
segments.
These pools were subjected to selection between cycles. Two final rounds,
cycles 5 and 6, of
"conventional shuffling," without addition of oligonucleotides, were performed
in an attempt
to further evolve the hybrid proteins.
Individual clones surviving later cycles were screened for improvement by
treating them with a single 40 ug/ml dose of MNNG and comparing survival to
untreated
samples. The best performing clone, from cycle 4, showed a 10-fold improvement
over the
wild-type at this dose. Its deduced protein (amino acid) sequence, shown in
Figure 5 (SEQ
ID N0:2), based on the improved (evolved) nucleotide sequence (SEQ ID NO:1 ),
contains 7
amino acid differences from the wild-type human alkyltransferase (see the
seven circled
amino acid residues in Figure 5}, 5 of which are found in other mammalian
alkyltransferases
(boxed residues in Figure 4). These 5 amino acid changes presumably were
encoded by the
oligonucleotides spiked in during cycles 3 and 4. All 5 were encoded by the
same degenerate
oligonucleotide pool, #7 in Figure 3. The other amino acid changes, Q (gln) to
R (arg) at
residue number 72 (Q72R) and G (gly) to D (asp) at residue number 173 (G173D)
(Taro
(1990) supra), were not present in the natural diversity and thus were created
by the
2 0 mutagenic shuffling process. In addition, 2 translationally silent
nucleotide changes (from the
wild type) were detected (see the two underlined nucleic acid residues in
Figure 5).
This shuffled mutant was characterized more thoroughly for its activity in E.
coli. In one set of experiments, cells were treated with graded doses of MNNG
and the
surviving fraction determined. Plasmids isolated from individual clones
surviving the
MNNG treatments were retransformed into GWR111. The retransformed clones were
screened individually by treating them with a single 40 ug/ml dose of MNNG.
The best
performing clone was further analyzed three ways: (i) The entire MGMT DNA
sequence was
obtained by sequencing the DNA target bidirectionally using fluorescent dye
terminator cycle
sequencing methods (Applied Biosystems 373A Autosequencer, Foster City, CA);
(ii) Kill
3 0 curves were established by treating exponentially growing cells with
graded single doses of
MNNG in the absence of isopropyl-beta-thiogalactopyranoside and measuring
colony-
71

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
forming ability relative to untreated controls. Cells harboring the wild-type
gene (pFC 14) or
the V 139F mutant (Christians ( 1996) Prod. Natl. Acad. Sci. USA, supra) were
treated in
parallel for comparison; (iii) The alkyltransferase activity of bacterial
extracts was
quantitated by in vitro exposure to calf thymus DNA containing O6-
[3H]methylguanine as
described in Bobola (1995) Molec. Carcinogen 13:70-80. Some extracts were
preincubated
with the mammalian alkyltransferase inhibitor O6-benzylguanine.
Survival was greater than for cells harboring either the wild-type human
MGMT or the V I39F mutant. The LD,o's, or dose of MNNG giving IO% survival,
were:
wild-type, 17.5 ug/ml; V 139F, 25 ug/ml; and cycle 4 shuffled mutant, 33
ug/ml. In a second
1 o set of experiments, bacterial extracts were exposed in vitro to an excess
of [3H]-methylated
DNA substrate, primarily in the form of O6-methylguanine, to measure total
alkyltransferase
activity. Average insoluble counts per minute per ug of total protein were:
wild-type, 126;
V 139F, 58; and cycle 4 shuffled mutant, 52. All three proteins were sensitive
to the inhibitor
C~-benzylguanine.
Thus, the recursive sequence recombination methods of the invention has
successfully generated a new and improved human alkyltransferase protein. The
random
diversity created by the mutagenic shuffling process was augmented by the
diversity provided
by nature. Natural diversity was utilized by simply mixing fragments of the
human gene with
oligonucleotides encoding all of the known mammalian amino acid substitutions.
Homology
2 0 to the human gene in the sequence flanking the regions of diversity
facilitated incorporation
of the oligonucleotides. The best performing mutant was a hybrid with 7 amino
acid
differences from the human alkyltransferase, as shown in Figure 5 (SEQ ID NO:1
). Two of
the mutations arose spontaneously during shuffling, and the other 5 were
encoded by the
natural diversity, specifically, one of the "spiked oligos" spanning amino
acid position 50.
2 5 Because all oligos were shown by PCR to be capable of hybridizing to the
human sequence, it
is likely that ali were incorporated into the pool at least to some degree.
Previous work with a different system also confirmed that synthetic oligos in
such a reaction are incorporated at approximately the expected ratios (Crameri
( 1996) Nature
Medicine, supra). Another way to incorporate natural diversity is to isolate
or synthesize the
3 0 cDNA from each of the species and shuffle the entire coding sequences
together. This
recursive method of breeding natural diversity will improve many related genes
from
72

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
different organisms as well as gene families within an organism. Furthermore,
it can be
applied to multiple proteins with related motifs, either structural or
functional.
It is difficult to mechanistically rationalize how the amino acid
substitutions in
the shuffled mutant increase its activity in E. coli. None of the amino acid
positions mutated
in the shuffled mutant was assigned a function in a computer model of the
human
alkyltransferase based upon the sole alkyltransferase crystal structure, that
of the bacterial
Ada protein C-terminal fragment (Wibley (1995) Cancer Drug Design 10:75-95;
Moore
( 1994) EMBO J. 13:1495-1501. The clustering of 5 of the mutations around
position 50 is
striking, but no known function has been ascribed to this region of the
protein. Three of these
5 substitutions are found in all of the other mammalian alkyltransferases.
While some
substitutions might be neutral, a possibility that can be answered by
backcrossing, others
might be synergistic, especially those involving charge changes. The proximity
of the G (gly)
to D (asp) mutation at position number 173 (G 173D) (see Tano ( 1990) supra,
for residue
numbering) to the conserved E (glu) at residue number 172 (E172) might be
significant given
the proposed involvement in crucial salt-link interactions by E172. An
additional acidic
residue in the region might enhance this effect.
The power of DNA shuffling is that it is a molecular breeding process that
allows for the combination of mutations which incrementally improve many such
complex
effects without having to model the effects in detail. We have exploited this
property to
2 0 evolve an alkyltransferase that is more potent in vivo than the natural
enzyme or any reported
mutants. This evolved mutant will be very useful in chemoprotection by gene
therapy. An
improvement over wild-type alkyltransferase is very useful to the clinician by
allowing dose
escalation of alkylating agents without the corresponding toxicity to the
patient. Once-
promising alkylating agents which are not used because of severe myelotoxicity
might now
become clinically acceptable. Even a slight improvement in alkyltransferase in
vivo is useful,
given that positive selection allows a relatively small number of resistant
cells to repopulate
the bone marrow. This alkyltransferase is further modified to incorporate
additional features
such as O6-benzylguanine resistance. The alkyltransferase can also be
subjected to additional
DNA shuffling and selected for additional improved activity in mammalian
cells, such as
3 0 improved nuclear localization, or better interaction with the eukaryotic
chromatin structure.
73

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
Example 4' Whole Genome Shuffling of Virus by In Vivo Recombination Using
Adenovirus
P a emi s
This example demonstrates the construction of an novel adenovirus-phagmid
using the recursive recombination methods of the invention which is capable of
packaging
DNA inserts over 10 kilobases in size. Incorporation of a phage fl origin
using the methods
of the invention also generates a novel in vivo shuffling format capable of
evolving whole
genomes of viruses, such as the 36 kb family of human adenoviruses.
The widely used human adenovirus type 5 (Ad5) has a genome size of 36 kb.
It is difficult to shuffle this large genome in vitro without creating an
excessive number of
changes which may cause a high percentage of nonviable recombinant variants.
To minimize
this problem and achieve whole genome shuffling of AdS, an adenovirus-phagemid
was
constructed using the methods of the invention.
As outlined in Figure 6, the 36 kb Ad5 genome was divided into two
overlapping parts by restriction digestion. Each of the two halves were
subcloned into
pBR322; the resulting two plasmids designated pAd-R and p-Ad-L. Specifically,
an EcoR I
ready-made adaptor was first ligated to each end of the linear 36 kb genomic
DNA. This
ligation product was then digested with BamH I to generate the right half of
the Ad5 genome
(nucleotide 21,562 to 35,935); and, with EcoR I to generate the left half of
the genome
(nucleotide 1 to 27,331). The right half 14.3 kb BamH I /EcoR I fragment was
then ligated
with BamH I /EcoR I digested pBR322 to create Ad-R, and the left half 27.3 kb
EcoR I
fragment was ligated with EcoR I digested pBR322 to created pAd-L. For gene
transfer and
safety reasons, the Ad5 E I region was subsequently deleted from the pAd-L by:
first, creating
an Afl II restriction site at nucleotide 455 using site directed mutagenesis
(changing G residue
at position 457 to a T residue, and a C residue at position 459 to an A
residue); and, Afl II
2 5 partial digestion was then performed since there are other Afl II sites in
the plasmid. The
24.3 kd Afl II fragment was gel purified, filled in with DNA polymerase I to
create blunt
ends. It was then ligated with a Swa I linker to simultaneously delete the E1
region between
nucleotides 458 and 3533, and insert a unique Swa I site for insertion of
foreign genes.
To construct phagemids ssDNA phage fl replication origin was obtained by
3 0 PCR from pBluescript II KS(-) phagemid (Stratagene, San Diego, CA) and
ligated into the
Cla I site of the Ad plasmids (pAd-R and pAd-L-1) by recombinant DNA
techniques, as
illustrated in Figure 6. The resulting Ad-phagemids were then introduced into
a mutator
74

CA 02268265 1999-03-24
WO 98!13485 PCT/US97/17302
strain mutDS (see Degnen ( 1974) J. Bacteriol. 117:477-487) to obtain
mutations, thus
increasing diversity. The spontaneous mutation rate of mutDS strains is
approximately 1.8 x
10-6/base pair/cell/generation (see Fijalkowska (1996) Proc. Natl. Aca. Sci.
USA 93:2856-
2861 ), which is about 100 fold lower than that of in vitro shuffling (see
Stemmer ( 1994)Proc.
Natl. Aca. Sci. USA 93:2856-2861 ).
To prepare phagemid phage, these mutated Ad-phagemids were purified from
the mutDS cells and then introduced into a F+ recAl strain (XL-1 Blue,
Stratagene, San
Diego, CA), and the resulting transformants were infected with a helper M 13
phage
(VCSM13, Stratagene, San Diego, CA) with a multiplicity of infection (MOI) of
10. The
to recAl mutation, which abolishes the recombinase activity of RecA (see Clark
(1965) Proc.
Natl. Aca. Sci. USA 53:451-459), is essential for the stability of the 29 kb
pAd-L-fl during
helper phage infection. Stable, high titer (> 10 ~ ° transducing units
per ml) stocks of Ad-
phagemid phage were obtained. These ssDNA phages carrying the Ad genome were
then
used to infect a mutS 201:Tn5 strain (see Siege! (1982) Mutat. Res. 93:25-33)
at high
multiplicity to promote recombination in vivo. Homologous recombination is
particularly
efficient between single-stranded forms of intracellular DNA. After
replication, the
phagemids within the cell behave as regular plasmids and undergo additional
plasmid-
plasmid recombination during subsequent cell propagation. The shuffled Ad-
phagemids were
finally recovered and purified from the cells, and used to transfect HeLa
cells to generate high
2 0 titer libraries.
Phagemid vector have been widely used for peptide display, cDNA cloning
and site-directed mutagenesis (see Mead (1988) Biotechnol. 10:85-102 for
review).
However, phagemid vector have not been used with large sizes (inserts) of DNA.
Conventional phagemid systems have not been used for cloning DNA fragments
larger than
2 5 10 kilobases or to generate large-sized (> 10 kb) ssDNA. The invention's
Ad-phagemid has
been demonstrated to accept inserts as large as 15 and 24 kilobases and to
effectively generate
ssDNA of that size. In a further embodiment, larger DNA inserts, as large as
50 to 100 kb are
inserted into the Ad-phagemid of the invention; with generation of full length
ssDNA
corresponding to those large inserts. Generation of such large ssDNA fragments
provides a
3 0 means to evolve, i. e. modify by the recursive recombination methods of
the invention, entire
viral genomes. Thus, this invention provides for the first time a unique
phagemid system

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
capable of cloning large DNA inserts (>10 KB) and generating ssDNA in vitro
and in vivo
corresponding to those large inserts.
Example 5 The generation of retroviral vectors carving mutant drug trans
orters
A pool of cells expressing a library of variants of ABC transporters is
generated by shuffling the wildtype cDNA such as e.g. the MDR1 or cMOAT cDNA
as
described for the MGMT gene in example 3. The libraries are cloned into a
retroviral
backbone such as described in PCT/NL96/00195 (filed May 7 1996 published under
W096/41875) followed by transfection into a retroviral packaging cell line.
After stable or
transient transfection flow cytometric sorting of cells pumping out the drug
most efficiently is
performed to rapidly select for those cells expressing the desired phenotype
from the
retroviral construct. If the MDR or cMOAT drug/substrate is fluorescent by
itself such as in
the case of anthracyclins or rhodamine for MDR1 this can be used to sort cells
expressing a
desired mutant. In the case of MDR1, fluorescent analogues of AZT (3'-azido--
2',3'dideoxythymidine), ddC (2',3'-dideoxycytidine) or etoposide, or BODIPY
conjugates of
paclitaxel (a taxol equivalent) can be used to viably sort or separate cells
negative for these
dyes from cells positive for the fluorescent drug and thus negative for a
particular MDR1
variant. Optionally flow cytometric sorting is followed by selection for those
cells actually
resistant to the drug used for flow cytometric sorting or direct cloning by
single cell sorting or
2 0 convential limiting dilution in tissue culture. Because the cells are
retroviral packaging cell
lines the selected cells can than be tested for the production of retrovirus
carrying the mutant
version of the ABC transporter under investigation.
An alternative to making recursive recombination libraries from single drug
resistance sequences is to subject a complete vector carrying for example MDR1
or cMOAT
2 5 to recursive recombination. This could be advantageous because the
performance of for
example a retroviral vector carrying a transgene such as MDR1 is influenced by
the transgene
polynucleotide sequence itself. Therefore optimal vectors for a given
application may be
generated by starting from a complete vector including but not limited to the
MDR1 retroviral
vector disclosed in PCT/NL96/00195.
76

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
F~xamnle 6~ Testing of selected pools of vectors cam mutant drug resistance
genes on human
hematonoetic stem cells b~flow c~ omet
Vectors generated using the methods disclosed herein that carry mutant drug
transporter genes are tested for their performance in stem cells by employing
flow cytometric
assays.
A mufti-color flow cytometric assay enables one to study multiple parameters
such as
differentiation, cycling, amphotropic receptor expression and retroviral
vector-mediated
transduction concomitantly at a single-cell level using immunophenotyping. The
most
primitive hematopoietic progenitors to study are the CD34b"g"'Lin(CD33, CD38
and CD71 )-
cells. This candidate hematopoietic stem cell population is identified by
staining with
monoclonal antibodies, conjugated to two different fluorochromes, and analysed
on two
emission channels. Another emission channel is used to measure transport
activity of mutant
drug transporter carried by a recombinant viral vector. Using such a
multiparameter flow
cytometric analysis drug resistance phenotype of selected MDRI or cMOAT or
MRP1 or
MRP3 variants are determined on CD34+lin- cells from human bone marrow or
human cord
blood cells or human peripheral blood cells. Variants exhibiting significant
transport activity
in CD34+Iin- cells are tested in vivo NOD-SCID mice (see example 7).
Example 7~ Testing of selected pools of drug~~esistance genes on human
hematopoetic stem
2 0 cells in vivo
After human patients and non-human primates, the NOD/Scid-Human chimera
murine model is the most valid assay to study human primitive hematopoietic
cells (Dick et
al, Semin Immunol. 8 (4):197-206,1996). By analyzing bone marrow cells from
mice
transplanted with umbilical cord blood CD34+ cells once a month, high levels
of engraftment
2 5 and mufti lineage differentiation are observed as soon as 4 weeks after
transplantation
Verlinden et al, Blood. 88: 168.,1996). After 6 months, human granulocytes,
platelets,
lymphocytes and erythrocytes are found in both the murine bone marrow and
peripheral
blood.
CD34+lin- cells as described under example 6 are isolated using FACS and
3 0 infected ex vivo with vectors generated using the methods disclosed here
and that carry
mutant drug transporter genes. The infected cells are then infused into
irradiated NOD/Scid
mice followed by in vivo selection of the transduced cells using the drug by
which the mutant
77

CA 02268265 1999-03-24
WO 98/13485 PCT/US97/17302
drug resistance gene was isolated. Doing so the in vivo performance of the new
drug
transporter or drug transporter vector or both is assessed by measuring
selective outgrowth of
the human stem cells as compared to CD34+lin- cells transduced with vector
carrying the
wildtype drug transporter.
The foregoing description of the preferred embodiments of the present
invention has been presented for purposes of illustration and description.
They are not
intended to be exhaustive or to limit the invention to the precise form
disclosed, and many
modifications and variations are possible in light of the above teaching. Such
modifications
and variations which may be apparent to a person skilled in the art are
intended to be within
the scope of this invention. All patent documents and publications cited above
are
incorporated by reference in their entirety for all purposes to the same
extent as if each item
were so individually denoted.
78

CA 02268265 1999-09-27
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS: Maxygen, Inc.; Introgene B.V.
(ii) TITLE OF INVENTION: Methods for Optimization of Gene Therapy
by Recursive Sequence Shuffling and Selection
(iii) NUMBER OF SEQUENCES: 41
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Smart & Biggar
(B) STREET: Box 11560, Vancouver Centre, 2200-650 W. Georgia
Street
(C) CITY: Vancouver
(D) STATE: British Columbia
(E) COUNTRY: Canada
(F) ZIP: V6B 4N8
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,268,265
(B) FILING DATE: 26-SEP-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/037,742
(B) FILING DATE: 27-SEP-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Smart & Biggar
(C) REFERENCE/DOCKET NUMBER: 80323-51
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 624 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
78a

CA 02268265 1999-09-27
(ix) FEATURE:
(A) CDS
NAME/KEY:
(B) 1.. 621
LOCATION:
(D) INFORMATION: "improved
OTHER /note= human
6-O-met hylguanine-DNA
methyltransferase
(MGMT) utant"
m
(xi) CE ESCRIPTION:SEQ ID O:
SEQUEN D N l:
ATG GACAAG GATTGT GAAATG AAACGC ACCACA CTGGAC AGCCCT TTG 48
Met AspLys AspCys GluMet LysArg ThrThr LeuAsp SerPro Leu
1 5 10 15
GGG AAGCTG GAGCTG TCTGGT TGTGAG CAGGGT CTGCAC GAAATA AAG 96
Gly LysLeu GluLeu SerGly CysGlu GlnGly LeuHis GluIle Lys
20 25 30
CTC CTGGGC AAGGGG ACGTCT GCAGCT GATGCC GTGGAG GCCCCA GCC 144
Leu LeuGly LysGly ThrSer AlaAla AspAla ValGlu AlaPro Ala
35 40 45
ACC CCTGAG TTGCTC GGAGGT CCGGAG CCCCTG ATGCAG TGCACA GCC 192
Thr ProGlu LeuLeu GlyGly ProGlu ProLeu MetGln CysThr Ala
50 55 60
TGG CTGAAT GCCTAT TTCCAC CGGCCC GAGGCT ATCGAA GAGTTC CCC 240
Trp LeuAsn AlaTyr PheHis ArgPro GluAla IleGlu GluPhe Pro
65 70 75 80
GTG CCGGCT CTTCAC CATCCC GTTTTC CAGCAA GAGTCG TTCACC AGA 288
Val ProAla LeuHis HisPro ValPhe GlnGln GluSer PheThr Arg
85 90 95
CAG GTGTTA TGGAAG CTGCTG AAGGTT GTGAAA TTCGGA GAAGTG ATT 336
Gln ValLeu TrpLys LeuLeu LysVal ValLys PheGly GluVal Ile
100 105 110
TCT TACCAG CAATTA GCAGCC CTGGCA GGCAAC CCCAAA GCCGCT CGA 384
Ser TyrGln GlnLeu AlaAla LeuAla GlyAsn ProLys AlaAla Arg
115 120 125
GCA GTGGGA GGAGCA ATGAGA GGCAAT CCTGTC CCCATC CTCATC CCG 432
Ala ValGly GlyAla MetArg GlyAsn ProVal ProIle LeuIle Pro
130 135 140
TGC CACAGA GTAGTC TGCAGC AGCGGA GCCGTG GGCAAC TACTCC GGA 480
Cys HisArg ValVal CysSer SerGly AlaVal GlyAsn TyrSer Gly
145 150 155 160
78b

CA 02268265 1999-09-27
GGA CTG GCC GTG AAG GAA TGG CTT CTG GCC CAT GAA GAC CAC CGG TTG 528
Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Asp His Arg Leu
165 170 175
GGG AAA CCA GGC TTG GGA GGG AGC TCA GGT CTG GCA GGG GCC TGG CTC 576
Gly Lys Pro Gly Leu Gly Gly Ser Ser Gly Leu Ala Gly Ala Trp Leu
180 185 190
AAG GGA GCG GGA GCT ACC TCG GGC TCC CCG CCT GCT GGC CGA AAC 621
Lys Gly Ala Gly Ala Thr Ser Gly Ser Pro Pro Ala Gly Arg Asn
195 200 205
TGA 624
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 207 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp Ser Pro Leu
1 5 10 15
Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His Glu Ile Lys
20 25 30
Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu Ala Pro Ala
35 40 45
Thr Pro Glu Leu Leu Gly Gly Pro Glu Pro Leu Met Gln Cys Thr Ala
50 55 60
Trp Leu Asn Ala Tyr Phe His Arg Pro Glu Ala Ile Glu Glu Phe Pro
65 70 75 80
Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser Phe Thr Arg
85 90 95
Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly Glu Val Ile
100 105 110
Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Lys Ala Ala Arg
115 120 125
Ala Val Gly Gly Ala Met Arg Gly Asn Pro Val Pro Ile Leu Ile Pro
130 135 140
78c

CA 02268265 1999-09-27
CysHisArg ValVal CysSer SerGly AlaVal GlyAsn TyrSer Gly
145 150 155 160
GlyLeuAla ValLys GluTrp LeuLeu AlaHis GluAsp HisArg Leu
165 170 175
GlyLysPro GlyLeu GlyGly SerSer GlyLeu AlaGly AlaTrp Leu
180 185 190
LysGlyAla GlyAla ThrSer GlySer ProPro AlaGly ArgAsn
195 200 205
(2)INFORMATION FOR SEQID
N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..55
(D) OTHER INFORMATION: /note= "oligo Al"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
CCTTAAGGAG GGGAAAAATG GCCGAGAYTT GTAAAATGAA ACGCACCACA CTGGA 55
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..58
(D) OTHER INFORMATION: /note= "oligo A2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
78d

I
CA 02268265 1999-09-27
AAATGGACAA GGATTGTGAA CTGAAATACA WKGTGTTCGA CAGCCCTTTG GGGAAGCT 58
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..61
(D) OTHER INFORMATION: /note= "oligo A3"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
AAATGAAACG CACCACACTG SMCAGCCCTT TGGGGGCGAT RGAGCTGTCT GGTTGTGAGC 60
A
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..55
(D) OTHER INFORMATION: /note= "oligo A4"
61
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
TGGAGCTGTC TGGTTGTGAG CGGGGTCTGC ACRGTATAAA GCTCCTGGGC AAGGG 55
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
78e

CA 02268265 1999-09-27
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..58
(D) OTHER INFORMATION: /note= "oligo A5"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
AGCAGGGTCT GCACGAAATA CGGTTCCTCA GCGGGAAGAC GTCTGCAGCT GATGCCGT 58
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..58
(D) OTHER INFORMATION: /note= "oligo A6"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
AGCTCCTGGG CAAGGGGACG CCTARMWCTG ATCCCAMAGA GGTCCCAGCC CCCGCTGC 58
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
78f

CA 02268265 1999-09-27
(B) LOCATION: 1..61
(D) OTHER INFORMATION: /note= "oligo A7"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
CTGCAGCTGA TGCCGTGGAG GCCCCAGCCW SCCCTGAGKK GCTCGGAGGT CCGGAGCCCC 60
T 61
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..61
(D) OTHER INFORMATION: /note= "oligo A8"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
CGGTTCTCGG AGGTCCGGAG TCCCTGGTGC AGTGCGAAAC CTGGCTGAAT GCCTATTTCC 60
A 61
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..58
(D) OTHER INFORMATION: /note= "oligo A9"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
78g

CA 02268265 1999-09-27
TGCAGTGCAC AGCCTGGCTG SAWGCCTATT TCCRAGAGCC CGAGGCTATC GAAGAGTT 58
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..58
(D) OTHER INFORMATION: /note= "oligo A10"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
ATGCCTATTT CCACCAGCCC KCGGCTACCC CAGGGCTGCC CGTGCCGGCT CTTCACCA 58
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..43
(D) OTHER INFORMATION: /note= "oligo All"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
AGGCTATCGA AGAGTTCCCC TTGCCGGCTC TTCACCATCC CGT 43
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
78h

CA 02268265 1999-09-27
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..43
(D) OTHER INFORMATION: /note= "oligo A12"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
ACCATCCCGT TTTCCAGCAA GATTCGTTCA CCAGACAGGT GTT 43
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..46
(D) OTHER INFORMATION: /note= "oligo A13"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
AGGTTGTGAA ATTCGGAGAA AYGGTTTCTT ACCAGCAATT AGCAGC 46
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..43
78i

I
CA 02268265 1999-09-27
(D) OTHER INFORMATION: /note= "oligo A14"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
CAGTGGGAGG AGCAATGAGA ARCAATCCTG TCCCCATCCT CAT 43
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..61
(D) OTHER INFORMATION: /note= "oligo A15"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
TCATCCCGTG CCACAGAGTG ATCCGCAGCR ACGGATCCAT TGGCAACTAC TCCGGAGGAC 60
T 61
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..58
(D) OTHER INFORMATION: /note= "oligo A16"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
GCAGCAGCGG AGCCGTGGGC CACTACTCCG GAGGACAGGC CGTGAAGGAA TGGCTTCT 58
78j

I
CA 02268265 1999-09-27
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..61
(D) OTHER INFORMATION: /note= "oligo A17"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
GGCTTCTGGC CCATGAAGGC TYCCCGAMGA GGCAGCCAGC CTTGGGGAAG CCAGGCTTGG 60
G 61
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..61
(D) OTHER INFORMATION: /note= "oligo A18"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
GGTTGGGGAA GCCAGGCTTG TSTAAGGRCT TAGCTCTGAY TGGGGCCTGG CTCAAGGGAG 60
C 61
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 base pairs
78k

CA 02268265 1999-09-27
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..46
(D) OTHER INFORMATION: /note= "oligo A19"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
GGAGCTCAGG TCTGGCAGGG WCCCGGCTCA AGGGAGCGGG AGCTAC 46
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..55
(D) OTHER INFORMATION: /note= "oligo A20"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
TGGCAGGGGC CTGGCTCAAG YCATCGTTCG RGTCCTCGGG CTCCCCGCCT GCTGG 55
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
781

CA 02268265 1999-09-27
(B) LOCATION: 1..58
(D) OTHER INFORMATION: /note= "oligo A21"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
TCAAGGGAGC GGGAGCTACC ACGAGCCCCR AGCTTTCTGG CCGAAACTGA GTATGAAG 58
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 207 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..207
(D) OTHER INFORMATION: /note= "human 6-0-methylguanine-DNA
methyltransferase (MGMT)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Met Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp Ser Pro Leu
1 5 10 15
Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His Glu Ile Lys
20 25 30
Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu Val Pro Ala
35 40 45
Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln Cys Thr Ala
50 55 60
Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu Glu Phe Pro
65 70 75 80
Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser Phe Thr Arg
85 90 95
Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly Glu Val Ile
100 105 110
Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Lys Ala Ala Arg
115 120 125
78m

CA 02268265 1999-09-27
Ala Val Gly Gly Ala Met Arg Gly Asn Pro Val Pro Ile Leu Ile Pro
130 135 140
Cys His Arg Val Val Cys Ser Ser Gly Ala Val Gly Asn Tyr Ser Gly
145 150 155 160
Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly His Arg Leu
165 170 175
Gly Lys Pro Gly Leu Gly Gly Ser Ser Gly Leu Ala Gly Ala Trp Leu
180 185 190
Lys Gly Ala Gly Ala Thr Ser Gly Ser Pro Pro Ala Gly Arg Asn
195 200 205
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 210 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..210
(D) OTHER INFORMATION: /note= "rat 6-0-methylguanine-DNA
methyltransferase (MGMT)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
r
Met Ala Glu Ile Cys Lys Met Lys Tyr Thr Val Leu Asp Ser Pro Leu
1 5 10 15
Gly Lys Ile Glu Leu Ser Gly Cys Glu Arg Gly Leu His Gly Ile Arg
20 25 30
Phe Leu Ser Gly Lys Thr Pro Asn Thr Asp Pro Thr Glu Ala Pro Ala
35 40 45
Cys Pro Glu Val Leu Gly Gly Pro Glu Gly Val Pro Glu Pro Leu Val
50 55 60
Gln Cys Thr Ala Trp Leu Glu Ala Tyr Phe His Glu Pro Ala Ala Thr
65 70 75 80
Glu Gly Leu Pro Leu Pro Ala Leu His His Pro Val Phe Gln Gln Asp
85 90 95
78n

CA 02268265 1999-09-27
Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe
100 105 110
Gly Glu Met Val Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro
115 120 125
Lys Ala Ala Arg Ala Val Gly Gly Ala Met Arg Ser Asn Pro Val Pro
130 135 140
Ile Leu Ile Pro Cys His Arg Val Ile Arg 8er Asp Gly Ala Ile Gly
145 150 155 160
Asn Tyr Ser Gly Gly Gly Gln Thr Val Lys Glu Trp Leu Leu Ala His
165 170 175
Glu Gly Ile Pro Thr Gly Gln Pro Ala Ser Lys Gly Leu Gly Leu Ile
180 185 190
Gly Ser Trp Leu Lys Pro Ser Phe Glu Ser Ser Ser Pro Lys Pro Ser
195 200 205
Gly Arg
210
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 211 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..211
(D) OTHER INFORMATION: /note= "mouse 6-0-methylguanine-DNA
methyltransferase (MGMT)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Met Ala Glu Thr Cys Lys Met Lys Tyr Ser Val Leu Asp Ser Pro Leu
1 5 10 15
Gly Lys Met Glu Leu Ser Gly Cys Glu Arg Gly Leu His Gly Ile Arg
20 25 30
Leu Leu Ser Gly Lys Thr Pro Asn Thr Asp Pro Thr Glu Ala Pro Ala
35 40 45
780

CA 02268265 1999-09-27
Thr Pro Glu Val Leu Gly Gly Pro Glu Gly Val Pro Glu Pro Leu Val
50 55 60
Gln Cys Thr Ala Trp Leu Glu Ala Tyr Phe Arg Glu Pro Ala Ala Thr
65 70 75 80
Glu Gly Leu Pro Leu Pro Ala Leu His His Pro Val Phe Gln Gln Asp
85 90 95
Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe
100 105 110
Gly Glu Thr Val Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro
115 120 125
Lys Ala Ala Arg Ala Val Gly Gly Ala Met Arg Ser Asn Pro Val Pro
130 135 140
Ile Leu Ile Pro Cys His Arg Val Val Arg Ser Asp Gly Ala Ile Gly
145 150 155 160
His Tyr Ser Gly Gly Gly Gln Ala Val Lys Glu Trp Leu Leu Ala His
165 170 175
Glu Gly Ile Pro Thr Gly Gln Pro Ala Ser Lys Gly Leu Gly Leu Thr
180 185 190
Gly Thr Trp Leu Lys Ser Ser Phe Glu Ser Thr Ser Ser Glu Pro Ser
195 200 205
Gly Arg Asn
210
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 210 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..210
(D) OTHER INFORMATION: /note= "hamster 6-0-methylguanine-DNA
methyltransferase (MGMT)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
78p

CA 02268265 1999-09-27
Met Ala Glu Thr Cys Lys Met Lys Tyr Thr Val Phe His Ser Pro Leu
1 5 10 15
Gly Lys Ile Glu Leu Cys Gly Cys Glu Arg Gly Leu His Gly Ile Arg
20 25 30
Phe Leu Ser Gly Lys Thr Pro Ser Ser Asp Pro Lys Glu Ala Pro Ala
35 40 45
Ser Pro Glu Leu Leu Gly Gly Pro Glu Asp Leu Pro Glu Ser Leu Val
50 55 60
Gln Cys Thr Thr Trp Leu Glu Ala Tyr Phe Gln Glu Pro Ala Ala Thr
65 70 75 80
Glu Gly Leu Pro Leu Pro Ala Leu His His Pro Val Phe Gln Gln Asp
85 90 ' 95
Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe
100 105 110
Gly Glu Met Val Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro
115 120 125
Lys Ala Ala Arg Ala Val Gly Gly Ala Met Arg Asn Asn Pro Val Pro
130 135 140
Ile Leu Ile Pro Cys His Arg Val Ile Cys Ser Asn Gly Ser Ile Gly
145 150 155 160
Asn Tyr Ser Gly Gly Gly Gln Ala Val Lys Glu Trp Leu Leu Ala His
165 170 175
Glu Gly Ile Pro Thr Arg Gln Pro Ala Cys Lys Asp Leu Gly Leu Thr
180 185 190
Gly Thr Arg Leu Lys Pro Ser Gly Gly Ser Thr Ser Ser Lys Leu Ser
195 200 205
Gly Arg
210
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 181 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
78q

CA 02268265 1999-09-27
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..181
(D) OTHER INFORMATION: /note= "rabbit 6-0-methylguanine-DNA
methyltransferase (MGMT)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Met Asp Lys Thr Cys Asp Leu Lys Tyr Lys Thr Leu Ala Ser Pro Leu
1 5 10 15
Gly Ala Ile Glu Leu Ser Gly Cys Glu Arg Gly Leu His Ser Ile Arg
20 25 30
Leu Pro Gly Lys Lys Thr Pro Glu Ala Asp Pro Ala Glu Ala Pro Ala
35 40 45
Thr Pro Glu Gly Leu Gly Gly Pro Lys Arg Thr Pro Glu Pro Leu Val
50 55 60
Gln Cys Glu Ala Trp Leu His Ala Tyr Phe His Glu Pro Ser Ala Ile
65 70 75 80
Pro Glu Leu Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu
85 90 95
Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe
100 105 110
Gly Glu Val Val Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro
115 120 125
Lys Ala Ala Arg Ala Val Gly Gly Ala Met Arg Ser Asn Pro Val Pro
130 135 140
Ile Leu Ile Pro Cys His Arg Val Ile Cys Ser Ser Gly Ala Val Gly
145 150 155 160
Asn Tyr Ser Gly Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His
165 170 175
Glu Gly Ala Arg Lys
180
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 207 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
78r

CA 02268265 1999-09-27
(ii) MOLECULE TYPE: peptide
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Asp or Ala"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Lys or Glu"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Asp, Thr or Ile"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Glu, Lys or Asp"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Met or Leu"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Arg or Tyr"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Thr, Ser or Lys"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 11
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Thr or Val"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
78s

CA 02268265 1999-09-27
(B) LOCATION: 12
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Leu or Phe"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 13
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Asp, His or Ala"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 18
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Lys or Ala"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 19
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Leu, Met or Ile"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 22
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Ser or Cys"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 26
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Gln or Arg"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 30
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Glu, Gly or Ser"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 32
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Lys or Arg"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 33
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Leu or Phe"
(ix) FEATURE:
78t

CA 02268265 1999-09-27
(A) NAME/KEY: Modified-site
(B) LOCATION: 34
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Leu or Pro"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 35
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Gly or Ser"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 36
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Lys or Gly"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 37
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Gly or Lys"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 39
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Ser or Pro"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 40
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Ala, Asn, Ser or Glu"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 41
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Ala, Thr or Ser"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 43
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Ala or Pro"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 44
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Val, Thr, Lys or Ala"
78u

CA 02268265 1999-09-27
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:46
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Val or Ala"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:49
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Pro , Thr, Cys or
Ser"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:50
(D) OTHER ORMATION: /product= "OTHER"
INF
/note= "Xaa = Ala or Pro"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:51
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Ala or Glu"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:52
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Val, Leu or Gly"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:58
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Pro or Ser"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:60
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Met or Val"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:63
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Thr or Glu"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:64
(D) OTHER RMATION: /product= "OTHER"
INFO
/note= "Xaa = Ala or Thr"
78v

CA 02268265 1999-09-27
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 67
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Asn, Glu or His"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 71
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = His, Arg or Gln"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 72
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Gln or Glu"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 74
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Glu, Ala or Ser"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 76
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Ile or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 77
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Glu or Pro"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 78
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Glu or Gly"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 79
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Phe or Leu"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 81
(D) OTHER INFORMATION: /product= "OTHER"
78w

CA 02268265 1999-09-27
/note= "Xaa = Val or Leu"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:92
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Glu or Asp"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:111
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Val, Thr or Met"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:112
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Ile or Val"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:136
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Gly, Ser or Asn"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:149
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Val or Ile"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:150
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Cys or Arg"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:152
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note= "Xaa = Ser, Asp or Asn"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:154
(D) OTHER RMATION: /product= "OTHER"
INFO
/note= "Xaa = Ala or
Ser"
(ix)FEATURE:
(A) NAME/KEY:Modified-site
(B) LOCATION:155
78x

CA 02268265 1999-09-27
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = or Ile"
Val
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 157
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = or His"
Asn
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 162
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = or Gln"
Leu
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 163
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = or Thr"
Ala
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 174
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = Ile or Ala"
His,
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 175
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = or Pro"
Arg
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 176
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = Thr or Lys"
Leu,
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 177
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = or Arg"
Gly
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 178
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa =
Lys or Gln"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
78y

CA 02268265 1999-09-27
(B) LOCATION: 180
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = or Ala"
Gly
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 182
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = Ser or Cys"
Gly,
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 183
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = or Lys"
Gly
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 184
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = Gly or Asp"
Ser,
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 185
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = or Leu"
Ser
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 188
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = Ile or Thr"
Ala,
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 190
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = Ser or Thr"
Ala,
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 191
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa =
Trp or Arg"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 194
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa = Ser or Pro"
Gly,
(ix)FEATURE:
78z

CA 02268265 1999-09-27
(A) NAME/KEY: Modified-site
(B) LOCATION: 195
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Ala or Ser"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 196
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Gly or Phe"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 197
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Ala, Glu or Gly"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 198
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Thr or Ser"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 199
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Ser or Thr"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 200
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Gly or Ser"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 201
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Ser or Pro"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 202
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Pro, Glu or Lys"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 203
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Pro or Leu"
78aa

CA 02268265 1999-09-27
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 204
(D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa = Ala or Ser"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
Met Xaa Xaa Xaa Cys Xaa Xaa Lys Xaa Xaa Xaa Xaa Xaa Ser Pro Leu
1 5 10 15
Gly Xaa Xaa Glu Leu Xaa Gly Cys Glu Xaa Gly Leu His Xaa Ile Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Thr Xaa Xaa Xaa Asp Xaa Xaa Glu Xaa Pro Ala
35 40 45
Xaa Xaa Xaa Xaa Leu Gly Gly Pro Glu Xaa Leu Xaa Gln Cys Xaa Xaa
50 55 60
Trp Leu Xaa Ala Tyr Phe Xaa Xaa Pro Xaa Ala Xaa Xaa Xaa Xaa Pro
65 70 75 80
Xaa Pro Ala Leu His His Pro Val Phe Gln Gln Xaa Ser Phe Thr Arg
85 90 95
Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly Glu Xaa Xaa
100 105 110
Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Lys Ala Ala Arg
115 120 125
Ala Val Gly Gly Ala Met Arg Xaa Asn Pro Val Pro Ile Leu Ile Pro
130 135 140
Cys His Arg Val Xaa Xaa Ser Xaa Gly Xaa Xaa Gly Xaa Tyr Ser Gly
145 150 155 160
Gly Xaa Xaa Val Lys Glu Trp Leu Leu Ala His Glu Gly Xaa Xaa Xaa
165 170 175
Xaa Xaa Pro Xaa Leu Xaa Xaa Xaa Xaa Gly Leu Xaa Gly Xaa Xaa Leu
180 185 190
Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Arg Asn
195 200 205
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
78bb

CA 02268265 1999-09-27
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..42
(D) OTHER INFORMATION: /note= "oligo #1"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
GCATCCGAAT TCCTTAAGGA GGGGAAAAAT GGACAAGGAT TG 42
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..31
(D) OTHER INFORMATION: /note= "oligo #2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
CCGCTAAAGC TTCATACTCA GTTTCGGCCA G 31
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
78cc

CA 02268265 1999-09-27
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /note= "oligo #3"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
TCGAGCCCCA GGCCTCCGCA 20
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /note= "oligo #4"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
CCGGTGCGGA GGCCTGGGGC 20
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /note= "oligo #5"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
AAGAGCGCCC AATACGCAAA 20
78dd

CA 02268265 1999-09-27
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /note= "oligo #6"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
TAGCGGTCAC GCTGCGCGTA A 21
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..22
(D) OTHER INFORMATION: /note= "oligo #7"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
ATGCAGCTGG CACGACAGGT TT 22
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
78ee

CA 02268265 1999-09-27
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /note= "oligo #8"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
TACAGGGCGC GTACTATGGT T 21
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
Cys Cys His His
1
(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
Cys Cys Cys Cys Cys Cys
1 5
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
78ff

CA 02268265 1999-09-27
Cys Cys Cys His
1
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Cys Cys Cys Cys Cys Cys Cys Cys
1 5
78gg

Representative Drawing

Sorry, the representative drawing for patent document number 2268265 was not found.

Administrative Status

2024-08-01:As part of the Next Generation Patents (NGP) transition, the Canadian Patents Database (CPD) now contains a more detailed Event History, which replicates the Event Log of our new back-office solution.

Please note that "Inactive:" events refers to events no longer in use in our new back-office solution.

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Event History , Maintenance Fee  and Payment History  should be consulted.

Event History

Description Date
Inactive: IPC expired 2018-01-01
Inactive: IPC from MCD 2006-03-12
Inactive: IPC from MCD 2006-03-12
Inactive: IPC from MCD 2006-03-12
Inactive: IPC from MCD 2006-03-12
Inactive: IPC from MCD 2006-03-12
Inactive: IPC from MCD 2006-03-12
Application Not Reinstated by Deadline 2003-09-26
Time Limit for Reversal Expired 2003-09-26
Inactive: Abandon-RFE+Late fee unpaid-Correspondence sent 2002-09-26
Deemed Abandoned - Failure to Respond to Maintenance Fee Notice 2002-09-26
Letter Sent 2001-07-05
Inactive: Single transfer 2001-06-05
Extension of Time for Taking Action Requirements Determined Compliant 2000-07-27
Letter Sent 2000-07-27
Inactive: Extension of time for transfer 2000-06-27
Inactive: Office letter 1999-12-06
Inactive: Delete abandonment 1999-12-06
Inactive: Office letter 1999-10-26
Inactive: Delete abandonment 1999-10-20
Deemed Abandoned - Failure to Respond to Notice Requiring a Translation 1999-09-27
Deemed Abandoned - Failure to Respond to Maintenance Fee Notice 1999-09-27
Inactive: Correspondence - Formalities 1999-09-27
Inactive: Incomplete PCT application letter 1999-06-22
Inactive: Cover page published 1999-05-31
Inactive: Notice - National entry - No RFE 1999-05-14
Inactive: IPC assigned 1999-05-12
Inactive: First IPC assigned 1999-05-12
Application Received - PCT 1999-05-10
Application Published (Open to Public Inspection) 1998-04-02

Abandonment History

Abandonment Date Reason Reinstatement Date
2002-09-26
1999-09-27
1999-09-27

Maintenance Fee

The last payment was received on 2001-09-04

Note : If the full payment has not been received on or before the date indicated, a further fee may be required which may be one of the following

  • the reinstatement fee;
  • the late payment fee; or
  • additional fee to reverse deemed expiry.

Please refer to the CIPO Patent Fees web page to see all current fee amounts.

Fee History

Fee Type Anniversary Year Due Date Paid Date
Basic national fee - standard 1999-03-24
MF (application, 2nd anniv.) - standard 02 1999-09-27 1999-09-21
Extension of time 2000-06-27
MF (application, 3rd anniv.) - standard 03 2000-09-26 2000-09-07
Registration of a document 2001-06-05
MF (application, 4th anniv.) - standard 04 2001-09-26 2001-09-04
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
INTROGENE B.V.
MAXYGEN, INC.
Past Owners on Record
HELMUTH H. G. VAN ES
WILLEM P. C. STEMMER
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

To view selected files, please enter reCAPTCHA code :



To view images, click a link in the Document Description column. To download the documents, select one or more checkboxes in the first column and then click the "Download Selected in PDF format (Zip Archive)" or the "Download Selected as Single PDF" button.

List of published and non-published patent-specific documents on the CPD .

If you have any difficulty accessing content, you can call the Client Service Centre at 1-866-997-1936 or send them an e-mail at CIPO Client Service Centre.


Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Description 1999-03-24 78 4,566
Description 1999-09-27 111 5,375
Cover Page 1999-05-31 1 47
Drawings 1999-03-24 7 197
Claims 1999-03-24 2 63
Abstract 1999-03-24 1 58
Drawings 1999-09-27 7 193
Reminder of maintenance fee due 1999-05-27 1 112
Notice of National Entry 1999-05-14 1 194
Request for evidence or missing transfer 2000-03-27 1 109
Courtesy - Certificate of registration (related document(s)) 2001-07-05 1 112
Reminder - Request for Examination 2002-05-28 1 118
Courtesy - Abandonment Letter (Maintenance Fee) 2002-10-24 1 179
Courtesy - Abandonment Letter (Request for Examination) 2002-12-05 1 167
PCT 1999-03-24 12 493
Correspondence 1999-06-22 2 57
Correspondence 1999-09-27 41 1,172
Correspondence 1999-10-20 1 7
Correspondence 2000-06-27 2 62
Correspondence 2000-07-27 1 10
Fees 1999-09-21 2 65

Biological Sequence Listings

Choose a BSL submission then click the "Download BSL" button to download the file.

If you have any difficulty accessing content, you can call the Client Service Centre at 1-866-997-1936 or send them an e-mail at CIPO Client Service Centre.

Please note that files with extensions .pep and .seq that were created by CIPO as working files might be incomplete and are not to be considered official communication.

BSL Files

To view selected files, please enter reCAPTCHA code :