Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION OF THE INVENTION
RETROVIRAL RECOMBINATION ASSAYS AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to United States Provisional Patent
Application
Serial No. 60/143,015, entitled "Measuring Genetic Recombination in HIV",
filed July 9,
1999, and to United States Provisional Patent Application Serial No. 60/164,
626, entitled
"Translentiviral Vectors and Transduction of Eukaryotic Cells Therewith",
filed
November 10, 1999, each of which applications is herein incorporated by
reference in its
entirety including all drawings.
BACKGROUND OF THE INVENTION
The following description includes information that may be useful in
understanding the present invention. It is not an admission that any of the
information
provided herein is prior art, or relevant, to the presently claimed
inventions, or that any
publication specifically or implicitly referenced is prior art.
This application relates to retroviral gene therapy vector design, safety, and
quality
assurance.
Retroviruses are characterized by a unique replication strategy in which the
genome is RNA, and is reverse transcribed into a linear double-stranded DNA,
which is
subsequently integrated into a host cell genome. From this "provirus" is
spawned copies
of the retroviral genomic RNA for encapsidation into virion particles that are
then
exported out of the cell to start the viral life cycle anew. The virion
particles are
manufactured in the cell according to genetic specifications encoded within
the proviral
genome.
All known retroviral genomes contain three major coding domains that direct
virion production and replication: gag, which directs the synthesis of
structural proteins
that form the matrix, the capsid, and the nucleoprotein structures; pol, which
contains the
information for the reverse transcriptase and integrase enzymes; and env, from
which are
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derived the surface and transmembrane components of the viral envelope
glycoprotein.
An additional smaller coding domain present in all retroviruses is pro, which
encodes a
protease enzyme that is recruited to the inside of the virion to effect
maturation of fusion
polyprotein products therein. The lentivirus subfamily of retroviruses further
encodes pro,
which is a part of pol (PR, RT, IN).
Retroviruses may generally be divided into two classes: simple and complex,
depending on the organization and representation of the genome. Complex
retroviruses, in
contrast to simple retroviruses, encode additional accessory and regulatory
proteins that
may be derived from differentially spliced messages coming from the genome.
Complex
retroviruses may further be divided into additional subgroups, among which are
the
lentiviruses.
In nature, lentiviruses are known to cause disease, principally by killing or
inducing loss of function of specific cells and tissues. One of the most well
characterized
lentiviruses is HIV, the causative agent of Acquired Immuno Deficiency
Syndrome
(AIDS) in humans. A comprehensive description of different retroviruses, their
biology,
and genetic organization may be found in Coffin et al., eds., Retroviruses,
Cold Spring
Harbor Laboratory Press, New York (1997), of which the skilled artisan is
aware.
Man-made retroviral vectors including HIV-based lentiviral vectors exist and
offer
significant utility in being able to transduce a variety of nondividing tissue
cells and
sustain expression of transgenes in vivo. Akkina et al. (1996) J. Virol. 70:
2581-2585;
Naldini et al. (1996) Science 272:263-267. Such tissues include but are not
limited to
brain, liver, muscle, and hematopoietic stem cells. Lentiviral vectors in
particular hold
great promise for gene therapy, and clinical trials to evaluate their safety
and efficacy for
treating certain human disease are being considered.
However, despite the advances and the promise these vectors hold, safety
concerns
still exist. Replication incompetent retroviral vectors can potentially
recombine to form
replication competent retroviruses (RCRs). This possibility, especially in the
instance of
pathogenic lentiviruses, should be minimized to maximize safety. To date,
lentiviral
vector systems have partially addressed this issue by expressing the essential
viral genes
on separate genetic elements (so-called "split-function") vectors. Naldini et
al., supra;
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Kafri et al. (1999) J. Virol. 73:576-84; Wu et al. (1997) EMBO, vol. 16, no.
16, pp. 5113-
5122. Nevertheless, the possibility still exists that RCRs can form from these
split
function systems, and current in vitro cell culture and animal model systems
are limited in
their ability to monitor and evaluate this.
A retroviral assay system that can sensitively monitor genetic recombination
events is therefore needed.
SUMMARY OF THE INVENTION
The invention features methods, systems, and indicator cells that provide for
a
sensitive determination of retroviral recombination and/or the potential for
production of
RCR or the emergence in vivo of potentially pathogenic viral forms.
The recombinants can contain one or more retroviral genetic determinants,
e.g.,
gag, pol, env, tat, and /or rev, which can originate from one or more
packaging constructs
such as is generally used in split function retroviral packaging systems. Env
is usually
supplied in its own construct. Nucleic acids that encode the packaging
component of the
vector particles are generally not efficiently packaged into retroviral
virion. The principal
function of the packaging constructs) is to package/encapsidate and help
transfer a gene-
transfer construct. These packaging constructs supply in trans the necessary
structural and
enzymatic retroviral proteins. "SIN" vectors are self inactivating vectors
that have, for
example, deleted U3 sequence in the 3' LTR. Upon one round of replication,
these
changes are copied into both the 5' and 3' LTRs, thereby producing a less
transcriptionally
active provirus.
The invention can measure recombination events that unite one or more of the
necessary genetic determinants contained within a wide variety of packaging
and gene-
transfer constructs. The result is detected by the establishment of a new,
"recombinant"
structure that contains genetic elements or portions thereof that were
formerly found
separately in the packaging and gene transfer construct components of the
system. It is
possible, although less likely so in the instance of SIN vector use, that
these recombinant
nucleic acids can then be packaged and mobilized to other cells because the
recombinant
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nucleic acids contain, and thereby may encode, at least some of the necessary
components
for replication and mobilization.
Because the functional retroviral gag-pol genetic structure is required for
any type
of RCR or viral DNA mobilization, monitoring for its presence in the form of a
retroviral
genetic recombinant allows a means to assess the risk of a
retroviral/lentiviral vector
creating an RCR event. As such, the invention provides a basis to measure the
safety of
vector stocks for use in gene transfer by using gag and pol, and more
specifically gag-pol,
as a surrogate marker for RCR.
Thus, in a first aspect the invention features a method of detecting a
retroviral
genetic recombinant, preferably a recombinant having gag and pol functions
which can be
complemented by helper functions) supplied by or to the cell, and which helper
functions) facilitate propagation of the recombinant to permit the
recombinant's
detection. This detection can take place using any one of a variety of
biochemical,
diagnostic, and/or functional assays that can identify the recombinant.
Preferably, the
assay makes used of the gag and pol functions contained within the
recombinant, and a
marker gene to facilitate the detection.
By "gag and pol functions" is meant to either a gag-pol fusion arrangement or
to
the supply of the individual gag and pol elements separated from one another.
Some
retroviruses, e.g., lentiviruses are characterized by the former, and others,
e.g.,
spumaviruses, by the latter. By "gag function" is meant a viral packaging
function
sufficient to produce a virion or virus-like-particle that is capable of
encapsidating a
retroviral genome. Such gag function does not necessitate expression of the
entire gag
sequence (Accola and Gottlinger (2000) J. Virol. 74:5395). By "pol function"
is meant any
one of the activities (enzymatic or non-enzymatic) or combinations of
activities provided
by protease (PR), reverse transcriptase (RT), and integrase (IN). These
functions may be
provided by the expression of the entire pol gene (PR, RT, IN), or by separate
expression
of the individual components thereof.
By "retroviral genetic recombinant" (hereinafter "recombinant") is meant a
species
of retrovirus that has undergone a nucleic acid recombination event, e.g.,
between one or
more nucleic acid strands or constructs, e.g., packaging and vector constructs
of split
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function retroviral systems. The retroviral genetic recombinants of the
invention may, in
addition to, and as a consequence of being derived from the events described
above, also
contain man-made or artificial sequences, including heterologous sequences
such as
reporter/marker gene sequences and mutations introduced into coding and non-
coding
regions.
"Propagating" or "propagated" refers to the ability of the recombinant to
integrate
and duplicate in the host cell, e.g., as part of a mitosis event, and/or to
support viral
replication and mobilization, e.g., using various helper functions that are
supplied by or to
the host cell. "Helper functions" are used to help facilitate propagation of
the recombinant
when the recombinant is one that is incapable or inefficient at replicating,
packaging,
and/or infecting by itself Examples of helper functions include but are not
limited to the
retroviral env gene product and pseudotypes thereof, and/or the retroviral tat
and rev gene
products. Depending on the embodiment, various helper functions can be
provided
individually or in combination. Usually, although not necessarily, these
helper functions
will originate from a different nucleic acid strand in the cell relative to
the recombinant
strand, although each may ultimately share the feature of being integrated
into the host
genome. This is usually a consequence of the "split function" aspect of most
retroviral
systems that is directed to "disarming" viruses and promoting biological
containment and
safety.
"Providing a cell suspected of having said recombinant" embraces any manner in
which the introduction or establishment of such recombinant can occur,
including but not
limited to infection, transfection, transduction, and transformation, as those
techniques are
commonly understood. The recombinant may be formed in the cell itself, e.g.,
from
different or related genetic elements present on different nucleic strands, at
a step
upstream to this, e.g., in another cell from which the intact recombinant is
then transferred
to the present cell, e.g., via mobilization, or formed in a virion particle.
However, exactly
how the recombinant is formed is not intended to be limiting of the invention.
The term "mobilization" is broad. It can relate to the packaging and/or export
of a
nucleic acid sequence, usually to another cell. The nucleic acid sequence is
preferably
either a retroviral genetic recombinant nucleic acid sequence or a marker gene
flanked by
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sequences that facilitate its mobilization and expression, e.g., packaging
into a retroviral-
like particle, reverse transcription, integration, and promoter-specific
expression. The
packaged nucleic acid may be contained within a retroviral virion, virus like
particle, or
capsid structure, which may also be isolated and assayed, e.g.,
extracellularly as a viral
supernatant. Usually, although not always, the packaging of the nucleic acid
is facilitated
by a viral packaging signal. For transfer/mobilization into another cell the
nucleic acid
sequence will also preferably contain cis-acting signals that facilitate
reverse transcription
and integration. If the mobilized nucleic acid is the marker gene, the
flanking sequences
will also preferably contain a promoter for the expression of the marker gene.
The
promoter may be an inducible promoter or a constitutive promoter. A preferred
embodiment for the latter is the human phosphoglycerate kinase gene promoter,
"pGK".
See Michelson et al. (1985) PNAS 82:20, pp. 6965-6969 and GenBank accession
M11958.
By "determining the presence of said retroviral genetic recombinant" can mean
any
assay that can detect mobilization or a recombinant, including but not limited
to e.g.,
hybridization assays, i.e., fluorescent in situ hybridization (FISH), antigen-
detection (e.g.,
ELISA), polymerase chain reaction (PCR), genetic marker response/mobilization,
and/or
recombinant mobilization. One or more such assays can be used together or
independently in making the determination. The features of the individual
cells used in
the method, and at each step (where multiple steps requiring multiple,
sometimes different,
cells are undertaken), may vary depending on the precise embodiment. An
example is
illustrated by use of the claim term "optionally" which is meant to denote
that various
embodiments are possible, some which utilize (and therefore require) a marker
gene
present in the cell, and those that do not require such a marker gene's
presence. An
example of the latter is when the assay relies solely on antigen-detection,
PCR, or marker
or gene mobilization to a second cell that may or may not contain such a
marker.
The individual embodiments noted are not necessarily mutually exclusive, and
may
be combined in certain other embodiments. Thus, in some embodiments both the
marker
gene and one or more other ways of determining the presence of the recombinant
are used,
e.g., PCR and antigen-detection. In other preferred embodiments the marker is
used and
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must be present in the recited cell, e.g., to denote the presence of and/or
enrich for
recombinants from among a greater population of non-recombinants, e.g., in a
viral stock.
In preferred embodiments, the retroviral genetic recombinant is integrated
into a
cellular chromosome and propagated or capable of propagation along with the
cell, e.g., as
a provirus. The marker gene, if it is present in the cell, is preferably also
integrated.
While the recombinant may harbor any one of a number of retroviral sequences,
e.g., coding and/or cis-acting sequences, other sequences including
heterologous
sequences may also be included. Examples of retroviral coding sequences
include gag,
pro, rt, in, pol, gag-pol, gag-pro, tat, rev, env, vpr, vpu, and vi~ "Cis-
acting" sequences
include those that possess functions in addition to or separate from coding
sequences, e.g.,
promoters, enhancers, frame-shift signals, polyadenylation signals, primer
binding
recognition sites (PBRs), LTRs, portions thereof, etc. Heterologous sequences
may
include reporter genes and inducer or repressor molecules, e.g., that can
activate or repress
a marker gene.
Preferably, the recombinant contains at least a gag sequence and at least some
pol
sequence, or derivative sequences thereof. Derivative sequences may include
mutations
consisting of silent mutations, stop codons, deletions, and/or insertions. The
recombinant
may also consist, at least in part, of a synthetic sequence, e.g., one having
silent mutations
in one or more codons, or with stronger or weaker codon usage in the target
host than the
natural retroviral coding sequence. By "gag and pol functions" is not
necessarily meant
"functional gag-pol". The latter term denotes coding sequence function and
activity
whereas the former can denote inactive presence, e.g., when in mutated format
or when
otherwise incapable of being propagated in the absence of "functional gag-pol"
or
functional gag and functional pol.
In embodiments wherein a mobilization is used to determine the presence of the
recombinant, that mobilization may be of the recombinant itself and/or of a
marker gene.
For this, the marker gene preferably has a retroviral packaging sequence, a
promter, and
flanking LTR elements, like the recombinant. The promoter may be an inducible
promoter or a constitutive promoter.
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Mobilization may be fostered by the in trans supply of an env gene or
pseudotype
thereof. The env gene may have previously been introduced to the cell, but
preferably is
introduced immediately prior to a desired mobilization event, e.g., in the
preferred form of
a pseudotyping agent such as a VSV-G protein, however introduced.
"Pseudotyping" is a
commonly understood term of art used to denote the combining of heterologous
envelope
species and/or other retroviral components, which may allow for modified
function over
the wild-type retrovirus, e.g, the ability to infect or replicate in a broader
or narrower host
cell range.
In especially preferred embodiments, the marker gene is a selectable marker
gene,
preferably one that encodes antibiotic resistance, preferably resistance to an
antibiotic, and
most preferably resistance to the antibiotic puromycin. In other aspects and
embodiments,
the marker gene can be synonymous with a reporter gene, e.g., luciferase,
green
fluorescent protein (GFP), or B-galactosidase. Preferably the marker gene is
affixed to a
promoter, inducible or constitutive, that is used to drive expression of the
gene within a
host cell.
In a second aspect, the invention features a method for detecting a retroviral
genetic recombinant having gag and pol functions. The method includes
providing the
recombinant in a cell as described for the first aspect, and additionally
includes a marker
gene in the cell that can be mobilized in the presence of the recombinant and
one or more
helper functions as described previously to thereby facilitate detection of
the recombinant.
In one preferred embodiment, the marker gene and recombinant are integrated
into
the host genome, with each capable of expressing a gene. Preferably, the gene
expressed
by either, including the recombinant, may be detected using an assay. The
assay for
detecting the recombinant's expressed gene can include, but is not limited, to
FISH, PCR,
antigen detection, Tat transfer, Gag transfer, and nucleic acid or gene
mobilization.
In a preferred embodiment, the recombinant includes one or more retroviral
coding
and cis-acting sequences, with the coding sequences preferably encoding one or
more
members selected from the group: gag, pro, pol, gag-pol, gag-pro, and
derivatives thereof.
Derivatives may include mutations such as insertions, deletions, silent
mutations, and stop
codon introduction.
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In other embodiments, the recombinant has genetic elements that are used to
mobilize itself and/or another nucleic acid sequence, e.g., a marker gene
provided in the
host cell.
Preferably, helper functions as described above are used to help facilitate
propagation of the recombinant. These can include, for example, the retroviral
env gene
product and pseudotypes thereof, and/or the retroviral tat and rev genes.
Thus, various
helper functions can also be provided in combination, e.g., by providing each
of env, tat,
and rev where appropriate.
In preferred embodiments, the marker is, once again, integrated into the host
cell
genome, e.g, into one or more of its chromosomes. For this, the host cell is
assumed to be
a eukaryotic host cell, preferably a mammalian cell, and preferably one
capable of being
infected or otherwise supporting replication of the subject recombinant. This
may require
helper functions.
Preferably the marker gene is a selective marker gene, more preferably an
antibiotic resistance gene, and most preferably a gene encoding resistance to
the antibiotic
puromycin. The gene is capable of expression from a constitutive or inducible
promoter,
which is preferably provided and operatively attached. The marker gene is
further
preferably flanked by cis-acting sequences that provide for its encapsidation,
reverse
transcription, integration, and/or expression.
In another aspect, the invention features a method for detecting a retroviral
genetic
recombinant in a cell that also includes a marker gene that is responsive to
the presence of
the recombinant within that cell. The method includes measuring the response
of the
marker gene to thereby detect the recombinant.
In preferred embodiments, the marker gene response is the result of a specific
gene
product encoded by the recombinant. This gene product may be a retroviral
gene, e.g., tat
or rev, or else one that encodes a heterologous inducer or repressor protein.
In any event,
the gene product influences marker gene expression, preferably through the
presence of a
corresponding cis-acting promoter and/or other response elements.
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In preferred embodiments that may further build on one or more of the above
embodiments, the marker gene is also a mobilizable marker gene, meaning that
it can be
mobilized.
The marker marker gene can be a reporter gene or a selective marker gene.
Preferably it is a selective marker gene, more preferably an antibiotic
resistance gene, and
most preferably a puromycin resistance gene. The marker preferably has an
expression
that is controlled by a promoter that is present, whether inducible or
constitutive.
In another aspect, the invention features a retroviral assay system for
detecting a
retroviral genetic recombinant having gag and pol functions. The system
features
a cell suspected of containing the recombinant, propagation of which is
facilitated
in the presence of one or more helper functions. The system further features a
means for
detecting the recombinant. The means includes, but is not limited to
expressing a marker
gene that is also present in the cell. Preferably, the marker gene is
responsive to the
presence of the recombinant, preferably to a gene product encoded therein.
Additionally or
alternatively, the means for detecting the recombinant can include mobilizing
the marker
gene, mobilizing the recombinant; and/or assaying for a product encoded or
otherwise
produced by the recombinant or said marker gene.
Preferably, the recombinant derives from a retroviral vector, and more
preferably
from a lentiviral vector, which is a preferred embodiment for all the aspects.
The
lentiviral-based vector may include, but is not limited to, HIV-1, SIV, HIV-2,
FN, EIAV,
BIV, CAEV, and OVINE.
Preferably, as with the foregoing aspects, the one or more helper functions is
further included to allow or help propagate the recombinant. The helper
functions that can
be used include, but are not limited to, env and pseudotypes thereof.
Preferably, the means for detecting the retroviral genetic recombinant is the
expression of a marker gene that is responsive to the presence of the
retroviral genetic
recombinant. The means may further include detecting one or more genes
expressed by
the recombinant.
In preferred embodiments, the marker gene encodes a product that can be
selected
for under one or more environmental conditions. Preferably, the marker gene is
an
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antibiotic resistance gene, and more preferably one that encodes resistance
against
puromycm.
Preferred embodiments for the assay system include components that perform or
assist in performing PCR, FISH, antigen-detection, Tat transfer, Gag transfer,
and gene
mobilization, or any other biochemical assay.
In another aspect, the invention features an indicator cell for indicating the
presence of a retroviral recombinant, an RCR event, and/or a wild-type
retrovirus. The
cell has an integrated marker gene, preferably a selectable marker gene.
Preferably the
marker gene is responsive to the presence of one or more genetic elements
encoded by the
retrovirus, e.g., tat, or a heterologous inducer or repressor protein. The
marker gene may
also be capable of mobilization as described above, and to that end may
include cis-acting
elements suitable for accomplishing or facilitating such.
In some embodiments, the indicator cell may also include helper functions,
e.g.,
env or a pseudotype thereof, when the retrovirus is one incapable of
replicating,
packaging, and/or infecting by itself. Such helper functions may be
heterologously
engineered into the cell. In another embodiment, tat is built into the cell to
assist retroviral
expression, replication, and/or packaging.
The indicator cell is preferably an immortalized mammalian cell, more
preferably
one that is capable of propagating an animal retrovirus, preferably a
lentivirus, more
preferably a human lentivirus, and most preferably a human HIV lentivirus. The
indicator
cell can be HeLa, 293T, a hematopoietic stem cell, or a derivative thereof.
Most
preferably, the cell is a 293T cell.
Preferably, the selectable marker gene of the indicator cell is an antibiotic
resistance gene, preferably one that encodes a product that is resistant to
puromycin.
In another aspect, the invention features a method, retroviral assay system,
or
indicator cell of any of the preceding aspects and embodiments that is further
used to
evaluate the risk of producing a replication-competent retrovirus from a
retroviral vector.
For this, the starting product may be a retroviral recombinant, e.g., one
lacking one or
more functional members selected from gag, pro, rt, int, env, tat, rev, and/or
other gene(s).
By complementing the lacking function in trans, one can test the relative risk
of a vector
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for achieving replication-competence, especially in vivo. In this aspect, the
invention may
further feature a method, system, or cell used for determining the risk of
producing a
replication-competent retrovirus (RCR) from a test vector particle. This
aspect may
include any one or more of the embodiments described previously.
In one preferred embodiment, the method, system, or cell includes providing a
cell
with a selectable marker gene having an expression that is activated or
enhanced in
response to a protein encoded by a recombinant. The selectable marker gene
encodes a
product which can be selected for in a growth media for said cell under a
defined
environmental condition. The method, system, or cell may also include features
that
facilitate contacting the cell with a test vector particle that is potentially
capable of
producing the recombinant in the cell and then selecting for the expression of
the
selectable marker gene in the environmental condition to thereby determine the
risk of the
test vector particle producing a replication competent retrovirus.
As used herein, a "retrovirus" is any ribonucleic acid sequence that may be
reverse
transcribed into double stranded DNA and subsequently integrated into a host
cell
genome. Although the invention in principle applies to all retroviruses,
preferred
retroviruses and vector systems for use with the invention include the
lentiviruses, most
preferably those selected from the group consisting of HIV-1, SIV, HIV-2, FIV,
EIAV,
BIV, CAEV, and OVINE. The genomes for these lentiviruses, including the
locations of
the cis and trans elements contained therein, are all well known and available
in the art,
e.g., as found in GenBank. Although especially preferred for the invention is
the use of
HIV-1 (see Table l, below, for genetics), other systems may be similarly
designed, and
with similar effect. It is further anticipated that various designs of vector
systems having
heterologous coding and cis elements (combining elements from different
retroviral
species) may also be successfully employed using the various aspects of the
invention, and
implemented without undue experimentation.
By "replication-competent retrovirus " (RCR) is meant one that can replicate.
Three genes: gag, pol, and env are thought to be essential to wild-type
retrovirus
replication. One form of an RCR would include env on a common nucleic acid
sequence
with gag and pol. In addition, multiple "cis-acting" (noncoding) elements are
generally
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thought to be necessary, e.g., long terminal repeat (LTR) sequences, a
retroviral packaging
signal (~), a polyadenylation signal, a promoter or promoters for expressing
the genes
(usually within the 5' LTR sequence), a transfer RNA binding site (PBS), a
polypurine
tract (PPT) that facilitates initiation of first and second-strand DNA
synthesis, a repeated
R region that is required for transfer of DNA synthesis between templates; and
short,
partially inverted repeats that are located at the termini of the viral LTRs
and that are
required for integration. It is also possible that an RCR could be produced,
especially
through recombination in vivo, that does not contain env. Retrovirus particles
can be
pseudotyped with non-env protein that mediates infection. (See, e.g., Endres
et al.,
"Targeting of HIV- and SIV-infected cells by CD4-chemokine receptor
pseudotypes",
Science. 278:1462-1464; Mebatsion et al. (1997) "A CXCR4/CD4 pseudotype
rhabdovirus that selectively infects HIV-1 envelope protein-expressing cells",
Cell.
90:841-847; Schnell et al. (1997) "Construction of a novel virus that targets
HIV-1-
infected cells and controls HIV-1 infection", Cell, 90:849-857.) It is
conceivable that
cellular ligand-receptor interactions could mediate infection/entry or
retroviral particles.
As used herein, regardless of whether the RCR contains env on a single strand,
an RCR
will be symbolically depicted as LTR-~-gag-pol-env-LTR, although this is not
limiting to
the definition of RCR. When integrated into a host cell, this structure
constitutes one form
of a "provirus."
By "test vector particle" is preferably meant a retroviral virion, virus-iike
particle,
or retroviral vector particle bearing a nucleic acid construct that is
suspected of possessing,
or having the potential to generate, a recombinant in an infected host cell.
The test vector
particle may be present as part of a larger heterogenous population, and may
have a
minority representation in that population. The test vector particle is
preferably capable of
infecting a host cell. In preferred embodiments, that cell harbors a selective
marker gene.
Preferably, if the test vector particle is able to form a recombinant nucleic
acid species,
that species is the product of genetic recombination involving one or more
constructs, e.g.,
packaging and gene-transfer vector constructs. Preferably, the test vector
particle reflects
a potential recombination event between such constructs, and it is a purpose
of the assay to
test for this. It is not necessary that the packaging and vector constructs
from which the
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test vector particle derives have homologous sequences, e.g., gag coding
sequences. In
some embodiments, one or more mutations is present in the retroviral sequence
of a
recombinant, including but not limited to internal stop codons, silent
mutations and/or
deletions within the gag and gag-pol sequences of either of these constructs.
A "gene-transfer vector" or "gene-transfer construct" as used herein does not
require gag, pol, and env, and need only contain the "cis" signals noted
above, or
equivalents thereof, for vector packaging, reverse transcription, and
integration. Most or
all of the sequence encoding viral Gag, Pol, and Env can be absent and
replaced with other
sequences, e.g., those encoding a gene of interest such as a reporter gene. A
transfer
vector can be illustrated symbolically as LTR-~-seq-LTR.
By "recombinant provirus" is meant one that is produced by at least one
genetic
recombination event between one or more nucleic acid strands or constructs,
e.g.,
packaging and vector constructs of split function retroviral systems. Although
preferable,
such a recombinant may not be integrated into a chromosome of a cell.
A "risk of producing a replication-competent retrovirus" occurs by any
recombination event that produces a recombinant provirus. The preferred
purpose of the
assay is to measure the possibility for creation of LTR-~'-gag-pol-env-LTR (an
RCR), and
not necessarily its actual occurrence. Thus the assay is meant to serve, at
least in part, as a
means to assess the safety of administering a given vector (e.g., in gene
therapy
applications) to an animal, especially in the long term.
A "selectable marker gene" may be a reporter gene but is preferably a gene
having
an expressed product capable of affecting the growth characteristics of a cell
under one or
more environmental conditions. The expressed product can encode, for example,
a
product that endows resistance to an antibiotic. Examples include dhfr, which
confers
resistance to methotrexate (Miller et al. (1985) Mol. Cell. Biol. 5:431-437);
neomycin/hygromycin phosphotransferase, which confers resistance to 6418 and
hygromycin (Palmer et al. (1987) Proc. Natl. Acad. Sci: 84:1055-1059; Yang et
al. (1987)
Mol Cell. Biol. 7:3923-3928); and, most preferredly for the invention, a gene
that encodes
a product that is resistant to puromycin or phleomycin (Morganstern and Land
(1990)
Nucleic Acids Res. 18:3587-3596). As an alternative to antibiotic resistance,
the
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expressed product may overcome a metabolic deficiency of the host. Examples
include
hprt (Miller et al (1983) Proc. Natl. Acad. Sci. 80:4709-4713) and thymidine
kinase (tk)
(Shimotohno and Temin (1981) Cell 26:67-77; Wei et al (1981) J. Virol. 39:935-
944),
which allow host cells that are otherwise deficient for the functional genes
to grow in
S minimal media supplemented with, e.g., hypoxanthine and thymidine.
In preferred embodiments, the selectable marker gene is preferably integrated
into
the host cell genome, and is preferably flanked by one or more nucleic acid
sequences not
natural to the host cell it is in. Preferably the gene is flanked on its 5'
end by a retroviral
LTR sequence that may promote expression of the gene, and a retroviral
packaging signal,
e.g., LY. In most preferred embodiments, the gene is flanked on both sides by
an LTR or
SIN sequence that, under appropriate conditions, can be packaged, transferred
to other
host cells, and selected for under one or more environmental conditions.
In another preferred embodiment of the method, system, or cell, the selectable
marker gene is under control of an LTR promoter that is responsive to the
retroviral Tat
protein. The term "Tat" refers to a retroviral protein encoded by the
retroviral tat gene.
Tat activates transcription from the retroviral LTR promoter by binding to a
proximal
sequence, "TAR", that is located within the 5' LTR (the "R" region).
The terms "mobilized" and "mobilizing" as used herein denote the process,
method, or ability of replicating a recombinant and/or other provirus, e.g.,
one containing
a marker gene, and transmitting it to another cell. This may require helper
virus or
transfected genes that encode and express these products to supply helper
functions, e.g.,
packaging, virion production, and receptor binding capabilities (e.g., env).
In other preferred embodiments of the method, system, or cell, the recombinant
provirus includes a gag sequence that encodes a Gag protein. A "Gag" protein
is a
retrovirus structural protein that is processed into several smaller proteins
that form the
virion matrix, the capsid, and the nucleoprotein structures of a retrovirus.
Preferably, one
can detect Gag using one or more of several techniques common in the art,
e.g., as an
additional measure of determining the risk of producing a replication-
competent retrovirus
(RCR) from said test vector particle. These techniques include the use of the
polymerase
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chain reaction (PCR), antibody detection, e.g., as in a p24 assay, and/or
other biochemical
methods. These procedures and techniques are well understood in the art.
In still other preferred embodiments of the method, system, or cell, the
recombinant provirus includes a gag-pol sequence that can transcribe and
translate into
Gag, Pro, RT, and INT proteins. The term gag-pol refers to a continuous
nucleic acid
sequence that includes at least a portion of the gag, pro, rt, and int
retroviral genetic loci,
preferably in that order. By "functional gag-pol" is meant a nucleic acid
sequence that
encodes functional Gag, Pro, and Pol protein products which together can
promote the
packaging and mobilization of a retroviral and/or recombinant proviral nucleic
acid
sequence.
Preferably, a step is also incorporated in which the recombinant provirus'
presence
is determined using an inhibitor of reverse transcripase, e.g., Nevirapine.
In other preferred embodiments of the method, system, or cell, and included as
an
additional measure of determining the risk of producing a replication-
competent retrovirus
(RCR), is the step and/or machinery necessary for mobilizing the selectable
marker gene
to another cell in which the marker gene can also be selectably detected. This
embodiment may also include the use of an inhibitor of reverse transcriptase
such as
Nevirapine to identify that expression is derived from a recombinant gag-pol
provirus.
In other embodiments, the method, system, or cell detects env minus
recombinant
provirus. By "Env" or "env" is meant, respectively, the retroviral envelope
glycoprotein
and its corresponding genetic locus. Retroviral surface and transmembrane
components
derive from the envelope protein and are necessary for the production of
infectious
retroviral virion particles. An "env minus" provirus is one that lacks one or
more of these
capabilities.
In still other embodiments, the recombinant provirus protein that promotes
expression of the selectable marker gene is a Rev protein that functions by
virtue of its
ability to shuttle retroviral response element (RRE)-containing retroviral
transcripts from
the nucleus to the cytoplasm of a host cell for suitable expression. Thus, in
such
embodiments, the marker gene includes an RRE element that can interact with
the Rev
protein.
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In preferred embodiments, the test vector particle derives from a
recombination
event that occurs in a split function lentiviral packaging system, e.g., one
that includes at
least one packaging construct and a vector construct, as those constructs are
known in the
art. An "env construct" or pseudotyping equivalent may also be included. These
constructs, as explained above, typically supply in traps one or more of the
necessary gag,
pol, and env retroviral genes whose products are necessary for gene transfer.
The test
vector particle of the method, system, or cell may be present as a product of
an earlier
transfection event which yields a heterogenous population of virion
encapsidated retroviral
sequences, of which the test vector particle, if it represents or is to
produce a recombinant
provirus in an indicator cell of the assay method, system, or cell, may
represent but a
minority. A "test vector particle" may contain one or more nucleic acid
sequences that
constitute, or upon infection are capable of producing, a recombinant
retrovirus.
In preferred embodiments, the envelope construct may include a pseudotype
envelope sequence, such as derived from the G protein of Vesicular
Stomatavirus (VSV).
The packaging construct may further include a promoter and a retroviral
nucleic
acid sequence that encodes one or more products necessary for retroviral
packaging. The
promoter can promote expression of at least one of the retroviral gene
products necessary
for retroviral packaging, e.g., Gag, Pro, and Pol. The promoter can optionally
be an
inducible promoter, preferably selected from the group consisting of TK, RSV,
CMV, and
THE promoters, which promoters are known and widely available in the art for
use with
the invention.
In some embodiments, the vector construct may further include a gene of
interest,
e.g., a reporter or marker gene, that can be used to monitor the relative
success or absence
of recombination in the assay. Preferred reporter genes are selected from the
group
consisting of green fluorescent protein (GFP), luciferase, and B-galactosidase
(B-Gal).
The reporter may express from a promoter other than an LTR promoter, and may
be an
"inducible" or "constitutive" promoter as such promoters are known in the art.
Promoters
for use in the vector construct may include the same promoters listed above as
possibilities
for the packaging construct, e.g., the TK, RSV, CMV, and THE promoters
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In especially preferred embodiments, the selectable marker gene encodes
resistance to an antibiotic, e.g., puromycin., and the cell in which it is
provided is
preferably infectable with the test vector particle used, e.g., a lentiviral
based test vector
particle can infect a HeLa or 293T human cell, or a derivative thereof.
Preferably the cell
line used is an immortalized mammalian cell line. Preferably, the selection
can occur
within a period of time of two days less, and with a clear distinction of
"positives" from
"non-positives."
In another aspect, the invention features a marker rescue assay system for
detecting
and/or analyzing a recombinant. The system includes an "indicator cell" having
a
selectable marker gene whose expression is activated or enhanced in the
presence of a
recombinant provirus, and whose gene product can be specifically detected or
selected for
in a growth media for said cell under a defined environmental condition.
Preferred embodiments for this aspect may encompass any of those embodiments
already mentioned, or combinations thereof. For example, the recombinant may
be a
functional gag-pol recombinant in which the Gag and Pol retroviral gene
products are
expressed and can be transcomplemented with Env to result in packaging and
mobilization
of the marker gene, which is preferably integrated in the genome of the
indicator cell line.
In embodiments where the selectable marker gene is also present, it too can be
mobilized
and selected for in the cell it is mobilized to.
In yet another aspect, the invention features an indicator cell as noted
above. The
cell preferably has a marker gene, preferably a selectable marker gene,
operably linked to
a promoter. The marker gene exhibits altered expression in the presence of one
or more
proteins supplied, e.g,. from a recombinant provirus. In preferred
embodiments, the
proteins are retroviral proteins selected from the group consisting of Tat and
Rev.
Preferably, the selectable marker gene is flanked by LTR sequences and can be
mobilized
from said cell in the presence of functional Gag and Pol gene products. Env or
pseudotypes thereof may also be used to accomplish this.
In a preferred embodiment, the indicator cell line contains a stably
integrated copy
of the marker gene. The marker gene is preferably on a nucleic acid strand
that contains a
signal for packaging, i.e., a retroviral packaging signal.
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In another preferred embodiment, the indicator cell line may contain nucleic
acid
sequences for the expression of an env gene. The expression of env can
pseudotype gene
products of the recombinant provirus that contact the indicator cell line
allowing the
recombinant provirus to mobilize to another cell. Mobilization can be to
another env
containing cell or to other cell types. Mobilized recombinants can be detected
using a
variety of methods as described herein, including detection of gag gene
product, i.e., HIV-
1 p24 core protein.
A common theme that forms the basis for preferred embodiments of the indicator
cell systems of the invention is the generation of recombinants preferably
containing gag
pol. Such recombinant contains the minimal requirements, minus env and/or
another
helper function, that is necessary for replication and/or packaging.
Therefore, it is
principally gag-pol that the majority of assays, systems; and indicator cells
described
herein are intended to detect. However, Tat transfer and other marker transfer
assay
aspects may not require this.
Features of the claimed indicator cells may further include any use, feature,
or
combination of uses of features recited for the methods and system aspects.
In still a further aspect, the invention features a Tat transfer assay in
which an
indicator cell of the type described above is used. The indicator cell
includes a selectable
marker gene that is expressed in response to a retroviral Tat protein encoded
by a
recombinant provirus, and wherein the selectable marker gene encodes a
product,
preferably a product endowing puromycin resistance, which can be detected or
selected for
in a growth media for the cell under a defined environmental condition,
preferably in two
days time or less.
In another aspect, the invention features a Gag transfer assay that includes
an
indicator cell having a selectable marker gene. The selectable marker gene is
expressed in
response to a retroviral protein encoded by a recombinant provirus. The
recombinant
provirus includes functional gag and pol genes that are capable of producing
functional
Gag and Pol proteins. The selectable marker gene encodes a product which can
be
selected for in a growth media for the cell under a defined environmental.
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In yet another aspect, the invention features a marker mobilization assay that
includes an indicator cell. The indicator cell contains a selectable marker
gene that is
expressed in response to a protein encoded by a recombinant provirus. The
marker gene
can be mobilized from the indicator cell to another cell in the presence of
functional Gag,
Pol, and Env retroviral gene products. The marker gene encodes a product which
can be
selected for in a growth media for the cell under a defined environmental
condition.
Preferred embodiments for the Tat transfer, Gag transfer, and marker
mobilization
assay aspects may include any embodiment or combination of embodiments noted
for any
of the aspects that is consistent with a useful purpose. Further, the
method,system, and
indicator cell embodiments may combine any of the other aspect embodiments
described
herein as appropriate to a purpose and practice within the broad spirit of the
invention.
Advantages of the invention include the ability to detect retroviral genetic
recombinants in sensitive fashion, and the use of gag-pol as a surrogate
marker for
identifying RCR potential. The invention further affords improved safety and
quality
1 S control in the design, production, and implementation of existing and
future retroviral
vector systems, preferably in prelude to the use of such vectors in gene
therapy. The
invention is especially well-suited in assaying for recombinant provirus
produced from
packaging constructs in which gag-pol has been disarmed, e.g., through the
inclusion of
mutations and/or deletions.
Other advantages, aspects, and embodiments will be apparent from the figures,
the
detailed description, and the claims.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows an exemplary retroviral genome (HIV-1) having genetic components
that can be used in conjunction with the methods, assay systems, and indicator
cells of the
invention. See Table 1 for more details.
Fig. 2 shows an embodiment of a split function lentiviral vector assay system
having a packaging construct (pTRE-gag-poly, a vector construct (pHR-CMV-GFP),
and
an env construct (pVSV-G; also known as pMD.G).
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Fig. 3 shows how one aspect of the invention can significantly improve on
existing
Tat-transfer assays by utilizing a novel indicator cell line bearing an
antibiotic resistance
gene (e.g., puro) that is capable of conveniently enriching for (or
"capturing") the desired
LTR-tat-LTR and other recombinations. The tat gene product is only expressed
in the
system when introduced in proviral form and is capable of specifically
activating the LTR-
driven antibiotic resistance gene, which can then be detected through routine
selection
means.
Fig. 4 shows how embodiments of the inventions can be used in tat transfer,
gag
transfer, and marker rescue/DNA mobilization assays to enrich for and detect
the
occurrence of retroviral genetic recombinants.
Fig. 5 shows a tat transfer assay embodiment that makes use of the sensitivity
and
enrichment provided by the specific indicator cell line HeLa-Puro. The figure
shows how
the desired tat-containing recombinant can be specifically selected for using
an internal
antibiotic resistance marker.
Fig. 6 shows a PCR assay embodiment that may be performed to further verify
the
presence of a tat-containing recombinant.
Fig. 7 shows a gag transfer assay embodiment that makes use of the sensitivity
and
enrichment provided by the specific indicator cell line HeLa-Puro.
Fig. 8 shows the results of a p24 antigen assay embodiment for the invention
that
demonstrates gag transfer and expression.
Fig. 9 shows a marker-gene rescue/DNA mobilization assay that makes use of the
sensitivity and enrichment provided by the specific indicator cell line HeLa-
Puro. Also
shown is the mobilization of the puro marker gene from the HeLa-puro cell into
another
cell.
Fig. 10 represents one prediction of how recombination between packaging and
gene transfer constructs may occur to produce an LTR-~'-gag-pol-LTR event.
Fig. 11 shows a PCR assay embodiment that can be performed to verify the 5'
recombination event that produces the LTR-~I'-gag-pol-LTR event.
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Fig. 12 illustrates a sequence confirmation that genetic recombination has
occurred
between the packaging and gene-transfer constructs. The sequence analysis also
illustrates
that the recombinants have an open gag-pol reading frame.
Fig. 13 shows a PCR assay embodiment that may be performed to verify the 3'
recombination event that produces the LTR-~I'-gag-pol-LTR event.
Fig. 14 shows sequence analysis of the 5' (Panel A) and 3' (Panel B)
recombinant
junctions of the recombinant retroviruses obtained and their corresponding
vector and
packaging construct constituents as depicted in Figs. 11 and 13. The figure
also illustrates
a sequence confirmation that genetic recombination has occurred between the
packaging
and gene-transfer constructs. The sequence analysis demonstrates recombinant
between
the 3' end of the vector construct and the polyA tract of the packaging
construct.
Fig. 15 demonstrates validation of tat transfer via recombinant provirus using
the
non-nucleoside reverse transcriptase inhibitor Nevirapine.
Fig. 16 demonstrates validation of gag transfer via recombinant provirus using
the
non-nucleoside reverse transcriptase inhibitor Nevirapine.
Fig. 17 demonstrates validation of marker mobilization via recombinant
provirus
using the non-nucleoside reverse transcriptase inhibitor Nevirapine.
Fig. 18 shows another split function vector (a translenti system) embodiment
that is
incapable of transferring functional gag-pol by virtue of abrogating the
translation of RT-
IN sequence from the packaging construct. This essentially "disarms" the RT
and IN
products downstream of pro that are part of the same mRNA transcript and
normally part
of the same fusion protein prior to post-translational modification and
maturation of the
independent proteins.
Fig. 19 shows that the Trans-lentiviral vector cannot transfer gag in a gag-
transfer
assay Confirmation of this is provided when Vpr-RT-IN is supplied in trans.
Fig. 20 shows 5' junction sequence for recombinant proviruses, demonstrating
the
preservation of a stop codon at the beginning of the RT coding region for the
translenti
system, whereas the rt (pol) gene in the lentiviral~lerived recombinants has
an open
reading frame (no stop codon).
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Fig. 21 shows a third generation lentiviral vector system in which the tat and
rev
genes are supplied on a separate construct.
Fig. 22 indicates that genetic recombination has taken place between the
vector and
packaging construct, since the puro marker gene is mobilized from the 293T-LTR-
puro
cell to the HeLa-tat cell. Panel A shows the assay overview using the third
generation
vector system. Panel B shows the plated/selected colonies under different
experimental
conditions.
Fig. 23 illustrates a lentiviral vector packaging construction embodiment in
which
expression of Gag-PR-RT-IN is regulated using an Internal Ribosomal Entry Site
(IKES).
See Example 10.
Fig. 24 illustrates a traps-lentiviral vector split packaging system
embodiment in
which Gag and Gag-PR-RT-IN are expressed from separate coding sequences to
mutate
the highly conserved stem-loop structure in both Gag and Gag-PR-RT-IN. This
does not
have deleterious effects on the formation of infectious particles. See Example
10.
Fig. 25 illustrates an MLV vector packaging construction embodiment in which
expression of Gag-PR-RT-IN is regulated using an Internal Ribosomal Entry Site
(IRES).
See Example 10.
Fig. 26 illustrates an MLV-based vector split packaging system embodiment in
which Gag and Gag-PR-RT-IN are expressed from separate coding sequences to
mutate
the highly conserved stem-loop structure in both Gag and Gag-PR-RT-IN. This
does not
have deleterious effects on the formation of infectious particles. See Example
10.
Fig. 27a illustrates Vpr-RT-IN expression plasmids containing 1-18 kb of
stuffer
DNA, e.g., derived from lambda DNA ("Vpr-RT-IN/ST") for identifying which will
still
efficiently provide RT and IN function in traps. See Example 11.
Fig. 27b shows how to humanize Vpr, RT and IN genes by PCR.
Fig. 27c shows the codon usage of highly expressed human.
Fig. 28a-d shows various embodiments for splitting the structure of Gag/Gag-PR-
RT-IN into two separate coding sequences: one that express Gag and another
that
expresses Gag-PR-RT-IN.
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Fig. 29 illustrates an MLV-based transfer vector that contains a DNA flat
sequence
derived from HIV-1. To facilitate initiating the central strand synthesis, the
PPT sequence
derived from MLV is inserted upstream of the cPPT sequence.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The basic components of traditional retroviral-based gene delivery systems
include
packaging, vector, and envelope elements that are usually supplied on separate
genetic
constructs to promote safety ("split-function"; see, e.g., Figs. 2,18 and 21).
The packaging
construct traditionally encodes the structural (gag) and catalytic (poi
proteins that are
necessary to generate an infectious particle. The vector construct typically
contains the
genetic material of interest that is to be transferred into the target cell,
along with a
packaging signal (~), a promoter for gene expression, and cis-acting sequences
necessary
for reverse transcription and integration such as the long terminal repeats
(LTRs) and a
primer binding site (pbs). The envelope (env) component, also supplied on a
separate
construct, mediates recognition and entry of the virus into the target cell
and is usually a
heterologous env protein such as the G protein from vesicular stomatitis virus
(VSV-G).
The retrovirus gag-pol structure is so highly conserved among retroviruses
that it is
likely required for any type of retroviral RCR and DNA mobilization event.
Therefore,
sensitive assays capable of detecting the de novo establishment or absence of
this entity in
vector-transduced cells would be useful. Such in vitro assays could help to
evaluate
retroviral/lentiviral vector safety and predict the risk associated with RCR
and retrovirus-
induced DNA mobilization in vivo. Such assays would be especially indicated
prior to use
in clinical trials with humans to ensure that vector stocks are free of the
potential to
generate gag-pol containing recombinants.
The Center for Biologics Evaluation and Research (CBER) of the Food and Drug
Administration (FDA) has established assay guidelines for screening retroviral
vector-
producing cell lines and supernatant for the presence of RCR. However, these
guidelines
have been established only for the safety of MLV-based retroviral vectors, and
include
specific PCR and immunological assays to monitor for evidence of replication-
competent
MLV. Guidelines have not been established for the monitoring of lentiviral
vectors.
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Thus, there is a need to develop assays for measuring the safety of lentiviral
vectors.
When applying, developing, and evaluating these assays, it is important to
keep in mind
that the safety of lentiviral vectors, particularly vectors based on HIV,
should be held to a
higher stringency as compared to the current retroviral-based vectors.
Three in vitro methods for assessing HIV-based lentiviral vector recombination
and RCR have already been developed. Naldini et al. (1996) Science 272:263-
267; Kafri
et al. (1999) J. Virol., vol. 73, pp. 576-84. These assays include: (1) tat
transfer, which
monitors for the presence of the HIV tat gene in the target cell; (2) gag
transfer, based on
measuring the HIV-1 capsid protein (p24Gag) in the medium of vector-transduced
cells;
and (3) Marker Rescue, which looks for the transfer of a marker gene from
vector-
transduced cells to other cells. Transfer of the tat and gag genes are used as
markers to
determine if recombination between the gag-pol packaging and gene transfer
vector
elements has occurred.
It was our hypothesis that the traditional assays used to monitor
recombination/safety are not sufficiently sensitive, and therefore are limited
for detecting
and analyzing genetic recombination. Therefore, we developed methods to
increase the
sensitivity for detecting genetic recombination. Our approach takes advantage
that any
RCR will require the gag-pol structure, and that we have demonstrated that it
is possible to
disarm gag-pol by separating Gag-pro from RT-IN. We developed a cell line that
through
the expression of an inducible marker gene can indicate when a recombinant is
formed and
integrated into a host cell chromosome including those containing gag-pol.
This markedly
improves the sensitivity in detecting genetic recombinants and allowed us to
enrich for the
recombinants in order to conduct detailed genetic and biologic analyses.
Detecting
recombinants not only provides a marker for evaluating the safety of
lentiviral-based gene
therapy vectors, but also provides the means to understand the nature of the
recombinants
at the genetic level, and additionally can help in the design of new vectors.
In designing and implementing our new assay system, we focused on monitoring
genetic recombination between the packaging and gene-transfer constructs that
yields
functional gag-pol (e.g., LTR-~-gag-pol-LTR) in transduced cells. An RCR event
by
comparison would be expected to be more unlikely since it would require, in
addition to
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the recombination necessary to generate an LTR-~-gag-pol-LTR, an additional
recombination to include a functional env gene with result that an LTR-~-gag-
pol-env-
LTR sequence is formed. The chances of generating an envelope-minus
recombinant
lentivirus would therefore be expected to be greater than the chances of
generating an
RCR event. Nevertheless, the former can be used as a surrogate predictive
marker for the
latter. Based on this predicate, we hypothesized that sensitive in vitro assay
systems
capable of directly detecting genetic recombination between the packaging
construct and
transfer vector in vector-transduced cells especially those containing gag-pol
could be
formulated to provide reliable endpoints (surrogate markers) for evaluating
safety and
predicting the emergence of RCR and retrovirus DNA mobilization in vivo.
Detecting the transfer of the tat, gag-pol and/or marker genes) is used to
determine
if recombination has occurred between the gene-transfer and packaging element
vectors.
Transfer of a marker gene from one cell to another requires that a functional
gag/gag-pol
recombinant structure (LTR-~Y-gag-pol-LTR) be formed in the vector-transduced
cells.
This enables the production of retroviral particles capable of packaging
either or both the
recombinant lentivirus genome (LTR-~-gag-pol-LTR) and the selective marker
gene (e.g.,
as LTR-~I'-marker-LTR). Such particles, if pseudotyped with VSV-G or other
envs, are
capable of mobilizing retroviral sequences, including those having marker
and/or tat, to
other cells where, in the case of the marker, it can be tracked (e.g., using
selective
conditions). The pseudotyped env may be provided in trans by using a cell line
capable of
expressing VSV-G or other env genes capable of pseudotyping the recombinant.
Example 1: Generation of HeLa-puro: A Tat-dependent Indicator
Cell Line Capable of Capturing Lentiviral Vector
Recombinants
To genetically analyze HIV-based lentiviral vector recombination, we developed
a
cell line which could be used to specifically select and enrich for
recombinant genomes
arising from recombination between the packaging and gene transfer vector
components.
In this "indicator" cell line, the puromycin N-acetyl transferase (puro) gene
was integrated
into a HeLa cell with its expression under control of the HIV-1 LTR. Because
the HIV-1
LTR is trans-activated by the HIV-1 tat protein, puro expression within the
cell is induced
when the tat gene becomes integrated into the host genome and expressed. With
tat being
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encoded on the gag pol packaging construct only, recombination between the
vector and
packaging elements would have to occur in order for tat to be transferred into
the HeLa-
LTR-puro cell. Expression of the puro gene would therefore lead to the
detection and
enrichment of only those cells that harbor the tat gene.
To construct the line, we used an HIV-based vector to deliver the puro gene
under
control of the HIV-1 LTR. The pHR-puro expression plasmid was constructed
using
commonly available starting materials by ligating a PCR amplified DNA fragment
containing the puromycin gene (derived from pPUR; Clontech Laboratories) into
the pHR-
CMV-lacZ plasmid (Naldini et al. (1996) Science 272:263-267) at the CIaI/XhoI
sites.
The vector stock encoding the puro gene was generated by cotransfection of
pTRE-gag-
pol, pVSV-G (Naldini et al. (1996) Science 272:263-267), and pHR puro into
293T cells
(ATCC). Culture supernatant was harvested 60 hours post-transfection and used
to
transduce a HeLa cell line (ATCC).
Because tightly regulated expression of the puro gene is in part dependent on
its
site of integration within the HeLa cell genome, it was important to select a
cell clone that
not only had a high level of puro expression in the presence of tat, but also
displayed a
low level of background expression in the absence of tat. To select for
candidate indicator
cell lines, individual cell clones were expanded in culture and screened for
tat-dependent
expression of the puro gene. Each clone was infected with a pseudotyped HIV-1-
derived
vector encoding the tat gene. One cell clone, henceforth termed HeLa-LTR-puro,
was
selected because it was able to grow and form colonies in culture media
containing
relatively high levels of puromycin (>30 ug/ml) following infection with the
HIV vector,
but displayed low level background resistance to puromycin (<0.5 ug/ml) in the
absence of
infection.
Using routine methodologies, one of skill in the art can readily create
equivalent,
and perhaps even superior, cell lines. However, for convenience, the HeLa-LTR-
puro line
noted herein was deposited with an International Depository Authority ()DA)
(American
Type Culture Collection ("ATCC"; 10801 University Boulevard, Manassas, VA
20110-
2209) on 30 June 2000 under conditions satisfying the Budapest Treaty for
biological
deposits in support of patent applications. (accession number to be assigned).
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Example 2: Vector Constructions
A three component lentiviral vector system is shown in Fig. 4. The component
vectors are constructed as follows: The pCMV-gag-pol packaging plasmid was
constructed by inserting an EcoRI/BamHI DNA fragment of pSG3 (coordinates 258
to
8384) into the pcDNA3.1(+) plasmid (InVitrogen) under control of the CMV
promoter.
The recombinant plasmid was then modified by introducing a 39-base pair
deletion in the
packaging sequence (~), and a 1357-base pair deletion in the envelope gene
(coordinates
5827 to 7184). The pHR-CMV-GFP plasmid was constructed by ligating a PCR
amplified
DNA fragment containing the GFP gene (derived from pEGFP-C1, Clontech
Laboratories)
into the BamHI/XhoI sites of the pHR-CMV-LacZ plasmid. Also, a 150 by sequence
of
DNA (coordinates 4327 to 4483) containing the central polypurine tract (PPT)
and central
terminal site (CTS) was PCR amplified from the HIV-1 pSG3 molecular clone and
ligated
into the unique CIaI site of pHR-CMV-GFP. The sequences in the vector
construct
illustrate the frameshift mutation that is used to introduce the premature
stop codon in the
gag gene. This was accomplished by the fill-in of a CIaI restriction enzyme
site that is
present at the S' end of the gag gene. The vector contains only the first 360
base pairs of
the HIV-1 gag coding sequence.
One of skill in the art can readily construct equivalent vectors using the
genetic
element delineations for the HIV-1 genome as reflected in Fig. 1 and Table 1
(based on
NCBI Genbank Genome Accession Number AF033819; SEQ. ID. No. 1). A more
detailed explanation of these components and their function may be found in
Coffin et al.,
of which the skilled artisan is aware. Those of skill will appreciate that
allelic variations
can exist between different isolates. In construction of the actual vectors
described herein,
isolates as described in Li et al. (1992) J. Virol. 66:6587 and Ghosh et al.
(1993) Virology
194:858 were used.
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Table 1: Genetic Elements and Coordinates of Human HIV-1
Genetic Element Coordinates
R: (1-96)
U5: (97-181 )
PBS: (182-199)
gag: (336-1836)
pro: (1637-2099)
pol: (2102-4640)
vif: (4587-5163)
vpr: 5105-5339)
tat: (5377-5591, 7925-7968
rev: (5516-5591, 7925-8197)
vpu: (5608-5854)
env: (5771-8339)
nef: (8343-8710)
PPT: (8615-8630)
U3: (8631-9085)
R: (9086-9181 )
Example 3: Preparation and Titration of Lentiviral Vector Stocks
Lentiviral vector stocks were produced by transfecting 5 ug of the pCMV-gag-
pol
packaging plasmid, 2 ug of the pVSV-G expression plasmid (also known as pMD.G;
Ory
et al. (1996) PNAS 93:11400) and 5 ug of the gene transfer (vector construct)
plasmid into
subconfluent monolayer cultures of 293T cells by the calcium phosphate DNA
precipitation method. Supernatants were harvested after 60 hrs, clarified by
low speed
centrifugation (1000g, 10 min), and filtered through 0.45-um pore-size filter.
The vector
particles were then subjected to ultracentrifugation using a Beckman SW-28
rotor (23,000
rpm, 90 min., 4°C) to concentrate vector particles. The pellets were
resuspended in 1.0 ml
of DMEM., aliquoted and frozen at -80°C.
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To determine vector titers, supernatant stocks of 1.0, 0.2, 0.04, 0.008,
0.0016, and
0.00032 u1 were used to infect cultures of HeLa cell, and GFP positive (green)
cell
colonies were counted two days later using a fluorescence microscope.
Example 4: Tat Transfer Assay
To improve on the traditional tat transfer assay, we developed the cell line
of
Example 1 that, through the expression of an inducible marker gene, can
indicate when a
recombinant is formed and integrated into the host cell chromosome (Figs 3-5).
Because
the HIV-1 LTR is trans-activated by the HIV-1 Tat protein, puro (puromycin N-
acetyl
transferase) expression is induced to confer resistance only if the cell
becomes infected
with a tat-containing recombinant that integrates and becomes expressed. Since
the tat
gene is present within the lentiviral packaging construct (linked to gag-poly,
recombinants
of the vector and packaging constructs would likely contain tat. Thus, these
integrated
recombinant lentiviruses (provirus) would express the Tat protein and
transactivate the
expression of the puro gene and confer resistance in puromycin-containing
media. This
novel tat transfer assay markedly improves the sensitivity for detecting
genetic
recombinants and importantly allows enrichment for the recombinants, making it
possible
for the first time to conduct detailed genetic and biologic analyses.
Utilizing our novel system we detected the 1000 resistant colonies derived
from
the 10e7 T.U. of lentiviral vector, indicating that the frequency of tat
transfer is about 10-4
to 10-5 (Fig. 5). The assay was performed as follows: 10~ i.u. of the
lentiviral stock was
used to infect 5x106 HeLa-LTR-puro indicator cells (MOI=2). As a control, the
indicator
cells were mock infected under identical conditions. Two days after infection,
cells were
trypsinized, split into five 100-mm tissue culture plates and cultured in
media containing
puromycin (5 ug/ml). After nine days of selection in puromycin containing
media, the
cells were fixed in methanol and stained with crystal violet. As shown,
puromycin
resistant colonies formed from HeLa-puro cells transduced with the lentiviral
vector stock
(approximately 1000 colonies per 10~ i.u.). In contrast, no colonies arose
from the mock-
infected culture. These results suggested that the tat gene was transferred
into the HeLa-
puro cell from the lentiviral stock.
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Although unlikely, the above results cannot exclude the possibility that the
tat gene
was transferred via plasmid carned over from the production of the vector
stocks in the
culture supernatants. To test this, HeLa-puro cells were infected with
supernatants from
the 293T transduced cells in the presence of Nevirapine (NVP), an inhibitor
specific to the
HIV-1 RT. NVP would inhibit only HIV-RT-mediated Tat transfer - but not if it
was
transferred through plasmid DNA. In the presence of NVP (25 ug/ml), no
puromycin-
resistant colonies were detected (Fig.lS). This result indicated that tat
transfer was
dependent on the HIV-1 RT, suggesting that the tat transfer was mediated by
recombinant
lentivirus. The presence of tat in the HeLa-puro cell's chromosomes was
confirmed by
PCR using primers specific to the first exon (219 bp) of the HIV-1 tat gene
(Fig. 6).
Example 5: Gag Transfer Assay
Because recombination can occur whereby only tat is combined with the gene
transfer vector construct, it was unclear from the tat-transfer results as to
whether
functional gag and pol genes were transferred as part of the recombinant
retrovirus into
1 S the HeLa-puro indicator cells. To determine if recombinants containing
functional gag and
pol genes were transferred, we developed a method of enriching for only those
recombinants that have open gag and pol reading frames (resulting from
recombination
between packaging and gene-transfer vectors). An overview of this approach for
detecting
and characterizing lentiviral vector recombination is depicted in Figs. 4 and
7. The assay
includes two steps. First, 293T cells (ATCC, Virginia, USA) are infected with
vector
stocks. It is in these infected cells that recombinants are likely to be
generated and stably
integrated. Such recombinant genomes can be expressed and mobilize retroviral
sequences, especially when helper functions are provided in trans, e.g., env.
Second, a
Tat-inducible antibiotic resistance marker gene (e.g., puro) in the HeLa-puro
indicator cell
line is used to capture recombinants which contain functional gag and pol
genes. Utilizing
puromycin selection, we are able to specifically select and enrich for cells
containing
recombinant retrovirus such as LTR-~-Gag-Pol-Tat/Rev-LTR. While this assay
design
has been termed gag-transfer, the results also demonstrate retroviral DNA
mobilization.
Specifics of the assay we performed were as follows: the lentiviral vector
stock
(10~ i.u., MOI=2) was first used to infect 293T cells. Similar to the
transduction of the
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HeLa-puro cell line, recombinants should also integrate into the genomes of
the 293T
cells, and those recombinants that contain an open gag and pol reading frames
would
express viral Gag and Gag-Pol proteins, produce progeny particles, and package
the
recombinant RNA genome. After two days, the transduced 293T cells were
transfected
with the pMD.G expression plasmid in order to pseudotype any progeny vector
particles
that may be produced. To increase production of progeny particles, a tat/rev-
encoding
expression plasmid was also transfected. Three days following transfection,
culture
supernatant was harvested and concentrated by ultracentrifugation. The
concentrated
supernatant was now used to transduce the HeLa-puro indicator cell line. The
transduced
HeLa-puro cells were placed under puromycin selection as described above
followed by
fixation and staining of the colonies that may have formed. When VSV-G was
supplied in
traps, 390 puromycin-resistant colonies formed. (Fig. 7)
Thus, lentiviral vector-derived recombinants could transfer functional gag and
pol
genes into its target cell (293T). This result suggested that the retroviral
recombinant
1 S genome can be mobilized from the transduced 293T cells to the HeLa-puro
cells if an
envelope was provided in traps. Again, no colonies formed when supernatants
from
mock-transduced 293T cells were used to infect the HeLa-puro cells.
Gag transfer was further confirmed by measuring the amount of HIV-1 capsid
protein (p24 antigen) released into the supernatant of the puromycin-resistant
HeLa-puro
cells. Fig. 8 Method: The 390 colonies which formed following selection in the
puromycin-containing media were trypsinized and replated into replica
cultures. One half
of the cells were transfected with a plasmid expressing tat and rev while the
other half was
mock transfected. Culture supernatants were collected 60 hours later and a
small amount
was aliquoted and frozen at -80°C. Meanwhile, the remaining supernatant
was
concentrated by ultracentrifugation as described above and resuspended in 100
u1 of
DMEM. Both the unconcentrated and concentrated supernatants were analyzed for
p24
antigen using an HIV-1 p24 antigen ELISA assay (CoulterInc.). Fig. 8 shows
that up to 95
ng/ml of CA could be detected in the culture supernatant. Moreover, we also
found a
significant increase (up to 10 fold) in capsid protein if the puro resistant
cells were
transfected with the tat expression plasmid, indicating that Gag expression is
linked with
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the HIV LTR promoter (Fig. 7). This result demonstrated gag and gag-pol
transfer and
mobilization of the retroviral recombinant genome from the transduced 293T
cells to the
HeLa-LTR-puro cells.
In all, Fig. 8 demonstrates that gag was transferred and expressed in the
vector-
transduced cell. The increase in p24 production with co-transfection of tat
suggested that
the gag gene was linked to the HIV-1 LTR, and provided additional evidence
that
recombination occurred between the gag-pol packaging construct and the LTR-
containing
gene transfer vector. Our results further show that the recombinants generated
by the
lentiviral vector system mobilized both tat and gag and pol genes from the
vector
transduced cells (293T) into the puro-HeLa cell when the envelope (e.g., as
pseudotype
VSV-G protein) was provided in trans (Fig. 9). This result indicates that the
lentiviral
vector can regenerate recombinants (such as LTR-~-gag-pol-tat/rev-LTR) in the
vector-
transduced cell, and that this envelope-deficient recombinant retrovirus can
be mobilized
into other cells when the envelope is provided in trans.
To confirm that Tat expression was a product of LTR expression and not of the
packaging vector, the reverse transcription inhibitor Nevirapine (NVP) was
used to
identify the source of expression. The specific HIV-1 RT inhibitor NVP blocked
the
mobilization of the recombinants from the vector-transduced cell to the HeLa-
puromycin
cell, indicating that the retrovirus DNA mobilization is specifically mediated
by HIV-1
gag/gag-pol (Fig. 16).
Finally, the high molecular weight DNA was extracted from the puro-resistant
cells using a WizardT""genomic DNA purification kit (Promega, Madison, WI).
DNA
fragments of the 5' recombinant virus region (from U3 to gag, ~2 Kb)(Figs. 11,
13,) and
3' recombinant virus region (from Tat to U3;~2.SKb)(Fig. 14, 15B,) were
amplified by
PCR, and the sequences contained therein determined.
The 5' region of the recombinant was amplified using sense primer homologous
to
a sequence located in the U3 region of the 5'-LTR (sequence: 5'-ccg gaa ttc
tgg cta act agg
gaa ccc act gc-3'; SEQ. ID NO. 2) with an antisense primer that hybridizes to
a sequence
located at the 3' end of the gag gene (sequence: 5'-cgc gga tcc tta ttg tga
cga ggg gtc get
gcc-3'; SEQ ID NO. 3). Amplification of a 2054 by DNA product (the predicted
size of
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the U3-gag fragment) can only occur if the vector and packaging elements have
combined
through genetic recombination. A PCR product of the predicted size was
amplified from
the lentiviral-derived puromycin resistant colonies. Both PCR amplified DNA
fragments
were cloned and sequenced. In 10 of 10 clones, sequence analysis of the 5' PCR
fragment
confirmed the presence of U3 R US (the 5' LTR), and the ~I' packaging signal
(from the
gene transfer vector) linked with the gag gene from the packaging construct
(Fig. 12).
Sequence analysis of the 3' PCR DNA fragment also revealed a genetic linkage
between the packaging construct and the 3' sequence of the gene transfer
vector (Fig. 14).
Interestingly, the recombinant genomes contained the poly A signal and the
poly A tract
derived from the packaging construct, indicating that recombination had
occurred between
the poly(A) tract of the packaging construct mRNA and the untranslated
sequence
immediately upstream of the 3' LTR of the vector during reverse transcription
(Fig. 14B).
These results collectively demonstrate that the novel recombination-dependent
retrovirus DNA mobilization (gag-pol transfer) assays can detect envelope-
minus
recombinant lentivirus that arise (derived from the packaging construct and
transfer
vector).
Example 6: Marker Mobilization Assay
One of the primary concerns associated with the formation of a recombinant
containing a functional gag pol is the potential of mobilizing DNA from one
cell to
another. Such mobilization would both perpetuate and augment the potential of
RCR.
Since the puro-containing RNA transcript in the HeLa-puro indicator cell
contains the
HIV-1 packaging signal, this RNA could be packaged into viral particles
derived from the
recombinant. Such particles, if pseudotyped with VSV-G, should be capable of
mobilizing the puro gene into another cell.
In the recombinant gag-pol-mediated marker gene mobilization assay (see Figs.
9,
17), we tested this. Specifically, we tested whether the recombinant
lentivirus (LTR-~-
gag-pol-LTR) could mobilize a marker gene (e.g., in LTR-~-puromycin-LTR) from
the
vector-transduced cell to another cell. We pooled the puromycin-resistant
colonies
resulting from the gag transfer experiment and co-transfected them with the
pMD.G and
tat/rev expression plasmids. Supernatant was harvested and used to transduce
HeLa-tat
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cells Felber et al., J. Virol. 64:3734; Wu et al., J. Virol. 69: 3389,
followed by selection of
puromycin-resistant colonies as previously described.
It seemed likely that the recombinant retrovirus particles could package both
recombinant lentiviral genomes (LTR-~-gag-pol-LTR) and lentivirus vector
genome
containing a marker gene (such as LTR-~-puromycin-LTR) (Fig. 9). Figs. 9 and
17 show
that the puro gene was mobilized by the recombinant. This transfer was
completely
inhibited by NVP (Fig. 17), indicating that the marker gene mobilization was
mediated by
HIV-1-based retroviral recombinants (gag-poly.
Taken together, these results indicate that this novel cell-based
recombination
assay enables the detection of envelope-minus recombinant lentivirus derived
from the
genetic recombination between the packaging and vector elements of the
lentiviral vector
system. The recombinants containing the functional gag-pol structure in the
vector-
transduced cells can integrate into the host genome, express viral proteins
(including Gag
and Gag-Poly, produce retroviral particles, encapsidate viral RNA, and
mobilize the viral
genome to new target cells when a helper function, e.g, env, is provided in
trans.
Example 7: Molecular Characterization of Recombinants
Assay recombinants can be predicted to have the structure indicated in Fig 10,
which shows the daughter recombinant molecule (LTR-~'-gag pol-tatlrev-LTR) and
parts
of the parent molecules from which is formed the recombinant.
To verify this, we selected for cells harboring a tat-containing recombinant
genome and expanded these cells in culture to characterize the nature of the
genetic
element responsible for transfer of tat into the HeLa-puro cell. Chromosomal
DNA from a
cell pool was isolated using the WizardTM genomic DNA purification kit
(Promega). If
recombination occurred between the vector and gag-pol packaging components,
then we
would predict that tat was transferred as part of an LTR-~I'-gag pol-tat-LTR
recombinant
genome. To confirm this, PCR was used as described in Example 5 to amplify the
recombinant from genomic DNA derived from the pool of puromycin-resistant HeLa-
puro
cells. Again, amplification of a 2054 by DNA product is only able to occur if
the vector
and packaging elements have combined through genetic recombination. This, in
fact, was
confirmed.
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Primers specific only to the pTRE-gag-pol packaging plasmid were used to
determine if the packaging plasmid DNA was contaminating the genomic DNA
preparations. The sequence of the 5' primer which hybridizes to the leader
region of the
CMV intermediate early promoter is 5'-gac ctc cat aga aga cac cg-3' (SEQ ID.
No. 4).
PCR failed to amplify a 1703 by product, demonstrating that plasmid DNA was
not
contributing to the amplification of the recombinant genome.
The 5' and 3' primers used to amplify the 5' (2054bp) PCR product also encoded
EcoRI and BamHI restriction sites, respectively. This allowed us to clone the
PCR
product into pUC 119 vector DNA and sequence the individual clones. (Figs. 12,
14A)
Our sequence analysis confirmed that the vector and packaging constructs had
joined
through recombination. Also, it appears that the recombination events on the
5' end
occurred through homologous sequences because of the sequence overlap between
the
vector and packaging components. Since only recombinants with open gag reading
frames are able to be transferred from 293T cells to the HeLa-puro indicator
cells all
recombinants sequenced (10/10) contained an open gag reading frame.
The 3' end of the recombinant was confirmed by PCR using a sense primer
specific to the 5' end of the tat gene (sequence: 5'-ccg gaa ttc atg gag cca
gta gat cct aga c-
3'; SEQ. ID. No. 5) and with an antisense primer that hybridizes to the R
region located in
the 3'-LTR (sequence: 5'-cgc gga tcc gca gtg ggt tcc cta gtt agcc-3'; SEQ. ID.
No. 6).
(Figs. 14, 15B, 24B) As shown, PCR products of various molecular weights were
amplified, with a predominant PCR product approximating 2500 by in length.
The 3' (2500 bp) PCR product was cloned into a pUCl9 vector DNA and
individual clones sequenced. (Fig. 14B) Interestingly, the junction between
the 3'-LTR
and the packaging construct contained a polyA sequence of various lengths.
Further
analysis of the sequence revealed that the polyA sequence was derived from the
mRNA
transcript of the pTRE-gag-pol packaging component. These results demonstrated
that the
polyA tract can be used to promote non-homologous recombination between the
vector
and packaging constructs.
Although it seemed unlikely, it was possible that the tat and gag genes were
transferred without recominbation (pseudotransduction), via the pTRE-gag-pol
packaging
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plasmid DNA carried over from the production of the vector stock. To test for
this
possibility, infections of the HeLa-puro cells were performed in the presence
of
Nevirapine, a non-nucleoside reverse transcriptase inhibitor specific to the
HIV-1 RT.
(Figs. 15-17) Nevirapine would prevent only HIV-RT mediated transfer of tat
and gag
into the HeLa-puro cell but not if it is transferred via plasmid DNA. These
figures show
the results of the tat transfer, gag transfer, and marker rescue assays which
were performed
as described before except that the infection of HeLa-puro was carned out in
the presence
of 1 ug/ml Nevirapine (NVP). No puromycin-resistant colonies formed in any of
the
assays, indicating that these transfers were mediated by HIV-1 reverse
transcriptase and
not through plasmid DNA contamination.
Example 8: Assay Comparing Gag-Pro versus Gag-Pol Vector Systems
We previously described a trans-lentivirus vector system based on a gag-pro
(not
gag-pol) packaging construct that promotes a "disarming" of lentiviral
recombinants by
eliminating production of functional reverse transcriptase (RT) and integrase
(IN) from the
traditional packaging vector. See United States Patent Application Serial No.
09/460,548.
These necessary products (RT and IN) are instead expressed on their own,
separate nucleic
acid vector fused, e.g., to vpr, a sequence that when expressed is capable of
promoting
packaging of the fusion protein. See Wu et al. (1997) EMBO, vol. 16, no. 16,
pp. 5113-
5122, and United States Patent Application Serial No. 09/460,548, filed Dec
12, 1999,
each of which is herein incorporated by reference.
We tested our new assay with our elegant trans-lentivirus system to see if we
could
produce recombinant lentivirus containing the functional gag-pol structure.
First, we tested the trans-lentiviral vector in the tat transfer assay, which
does not
require functional gag-pol. 293T cells were transfected with the four DNA
components of
the trans-lentiviral system (Fig. 18), and three days later the culture
supernatants were
collected. The vector particles were concentrated by ultracentrifugation,
resuspended in
DMEM, and titered by limiting dilution infection of the cell line, as
described above.
After nine day of culture in medium containing puromycin, approximately 800
resistant
colonies were enumerated using bright-field microscopy (Fig. 18). This result
suggests
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that the packaging construct of the traps-lentiviral vector can recombine with
the gene
transfer construct.
To determine whether the novel traps-lentiviral vector design would prevent
the
generation of a recombinant containing the functional gag-pol structure,
vector stocks of
the traps-lentiviral vector were also analyzed for gag-transfer/DNA
mobilization. Exactly
as described above for the lentiviral vector (Fig.l6), 10e7 infectious units
of trans-
lentiviral vector particles were used to infect 293T cells. Culture
supernatants were
collected and used to infect 293T cells as described above. Three days later
these culture
supernatants were collected and processed exactly as described above for the
lentiviral
vector. This analysis demonstrated that the traps-lentiviral vector is unable
to produce
resistant colonies, indicating added safety over the lentiviral vector. To
confirm that this
was the result of the separation of RT-IN (or RT and IN) from gag-pol, the
experiment
was conducted in parallel (Fig. 19) with an experiment in which the Vpr-RT-IN
fusion
protein was provided in the 293T infected cells (via transfection of the vpr-
RT-IN
expression construct). Under these conditions, approximately 300 resistant
colonies were
produced (Fig. 20). These results suggest that while recombination between the
vector
and packaging components can occur, the separation of RT-IN from gag-pol
prevents the
mobilization of the recombinant genome. Therefore, this approach disarms the
gag-pol
structure and represents a method for improving the safety of lentiviral
vector gene
therapy. It is also notable that in combination with assays to monitor/measure
recombination and /or DNA mobilization, the traps-lentiviral vector safety can
be quality
assured by in vitro testing.
In order to confirm that the puro resistant colonies that were produced by
complementation of the vector particles with the Vpr-RT-IN fusion protein were
indeed
RT-IN deficient, the resistant cells were expanded in culture and the high
molecular
weight DNA was extracted for genetic analysis (Fig. 20). This analysis
demonstrated that
that the RT-IN coding region was defective since S of 5 recombinants that were
analyzed
contained a translational stop codon (TAA) at the first amino acid residue of
RT, as
introduced into the traps-lentiviral packaging construct originally. By
comparison, 5 of 5
recombinants of the lentiviral vector contained an open RT-IN reading frame
(Fig. 20).
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Our biologic and molecular results confirmed that the novel cell-based assay
could
sensitively monitor the recombinant gag-pol in the vector-transduced cells.
Example 9: Third Generation (Tat-minus) Vector Systems
Third-generation lentiviral vector systems remove the tat and rev genes from
the
packaging construct. While tat is not needed at all, the rev gene is still
required and is
therefore supplied in trans from a separate genetic construct (Fig. 21 ).
Theoretically, this
reduces the possibility for an RCR event. To avoid the dependency of a
recombination
assay on the presence of tat as a part of the lentivirus trans-lentiviral
vector packaging
construct, we assayed tat minus (3rd generation) vector for DNA mobilization
in a tat-
independent manner.
Fig. 22 shows an overview of such an assay embodiment, as well as data
demonstrating that the puro gene in such an assay can be transferred into HeLa
Tat cells if
VSV-G is supplied. This is the identical concept behind the marker rescue
assay described
above.
For assaying the 3rd generation vector we found that, instead of HeLa-LTR
puro,
the 293T-LTR puro cell line improved sensitivity. 293T-LTR puro is constructed
in the
same way as HeLa puro. The same puromycin resistance gene is used and
positioned
downstream of an HIV-1 LTR, and the genetic transcript from which it is
expressed
contains an HIV-1 packaging signal that allows encapsidation into an HIV
particle. The
line 293T-LTR-puro was selected for its high expression levels of the puro-
cantaining
transcript. The more puro transcript available, the better the chance of it
being
incorporated into a viral particle for transfer into a cell, and the more
efficient the system
is.
Using routine methodologies, one of skill in the art can readily create
equivalent,
and, perhaps, even superior cell lines to 293T-LTR puro. However, for
convenience, this
line was deposited with an International Depository Authority (IDA) (American
Type
Culture Collection ("ATCC"; 10801 University Boulevard, Manassas, VA 20110-
2209)
on 30 June 2000 under conditions satisfying the Budapest Treaty for biological
deposits in
support of patent applications. (accession number to be assigned).
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The third generation (tat-minus) vector stock is used to infect the 293T-LTR
puro
cell. If one of the 293T-LTR puro cells harbors an LTR-~I'-gag-pol-LTR
recombinant, the
recombinant can express the viral Gag and Gag-Pol proteins and form a viral
particle
which can encapsidate the mRNA of the LTR-~I'-Ogag-RRE-puro-LTR construct.
Fig. 23
depicts and verifies this. Although the number of recombinants (LTR-LY-gag-pol-
LTR) is
fewer (52/10e7), the assay is sensitive enough to detect them.
Example 10: A Vector System Including Regulation of the Expression
Level of Gag-PR-RT-IN Using an Internal Ribosomal
Entry Site
Using the cell-based genetic recombination assay described above, we
demonstrated that a new lentivirus vector packaging construct designed by
splitting gag-
pol into two components (gag/gag-pro and Vpr-RT-IN) significantly improved the
biosafety of the lentivirus vector by preventing the regeneration of a
recombinant
lentivirus containing functional gag-pol. Since the gag-pol structure is
highly conserved
among all retrovirus, this structure must exist in any type of replicating-
competed
retrovirus derived from genetic recombination. By disarming the highly
conserved gag-
pol structure, we can improve the biosafety of all retrovirus-based vectors,
including
simple retrovirus-based vectors (such as MLV) and lentivirus-based vectors
such as HIV,
SIV, FIV, EIAV, B1V, Visna, CAEV, and Ovine lentivirus).
In this example, we removed the ribosomal frameshifting site in the retrovirus-
based vector packaging system, which is high conserved among all retroviruses
and
essential for retroviruses to maintain a well regulated ratio of Gag proteins
to Gag-Pol
proteins in infected cells for producing infectious particles. This design
splits the
expression of Gag/Gag-PR RT-IN into two separate coding sequences: one that
expresses
Gag and another that expresses the Gag-PR-RT-IN. Here, the production of a
fixed ratio
of Gag to Gag-PR-RT-IN is regulated by the internal ribosomal entry site
(1RES) derived
from the encephalomyocarditis virus (EMCV), rather than by the ribosomal frame-
shift
site. Since the translational initiation efficiencies of the different IRES
mutants differs
considerably, this allowed us to select different IRES mutants to regulate the
production of
Gag/Gag-PR-RT-IN at a fixed ratio, similar to the ratio of Gag/Gag-PR-RT-IN
derived
from the natural Gag/Gag-PR RT-IN structure. Importantly, this demonstrates
that one
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can design transvector packaging systems for all retrovirus-based vectors
since the
approach does not require virion-associated proteins (e.g., Vpr and Vpx).
A specific nucleotide motif, the slippery sequence, as well as a downstream
mRNA
secondary structure are essential for the production of the fixed ratio of Gag
to Gag-PR-
RT-IN (Fig. 23 for lentivirus vector; Fig. 25 for MLV vector). The disruption
of the
highly conserved stem-loop structure completely blocks the production of the
fixed ratio
of Gag to Gag-PR-RT-IN, which is essential to produce the infectious
particles.
By separating the expression of Gag and Gag-PR-RT-IN into separate coding
sequences, we are able to mutate the highly conserved stem-loop structure in
both Gag and
Gag-PR-RT-IN constructs without deleterious effects on the formation of
infectious
particles (Fig. 24 for a trans-lentiviral vector system; Fig. 26 for an MLV-
based vector).
Since the highly conserved element no longer exists in this system, safety is
now improved
at two different levels: one in that Gag and Gag-PR-RT-IN are split into two
separate
coding regions which should prevent the regeneration of recombinant retrovirus
containing the functional Gag-Pol structure; and two in that the novel
packaging system
completely removes the ribosomal frameshifing site which is essential for all
retrovirus to
maintain the ratio of Gag and Gag-PR-RT-IN during the production of infectious
particles.
It is likely that without this highly conserved element, any type of
recombinant retrovirus
would be unable to produce the infectious particles and replicate.
Since we express the HIV-1 Gag and Gap-PR-RT-INT polyproteins in two
separate mRNAs, and the expression of Gag-PR-RT-IN polyproteins is no longer
dependent on the highly conserved stem-loop structure of mRNA, this allows us
to
completely humanize all of the codons in the mRNA of Gag and Gag-PR-RT-IN.
Therefore, modified gag-PR-RT-IN genes would express high levels of protein to
produce
high titer vector.
Example 11: Preventing Regeneration of Recombinant Lentivirus Vector
Containing Functional Gag-PR-RT-IN
In current translentiviral packaging systems, there are large homologous
sequences
between the trans-enzyme plasmid (Vpr-RT-IN) and the packaging construct
(gag/gag
pro). If both mRNAs (trans-enzyme plasmid and packaging construct) are
nonspecifically
copackaged into the same virus particle, it is possible to regenerate the gag-
pol structure
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through homologous genetic recombination. There are two approaches to
preventing the
regeneration of the gag-pol structure. First, controlling the incorporation of
Vpr-RT-IN
mRNA into vector particles will eliminate the possibility of regenerating gag-
pol since the
retrovirus genetic recombination occurs within the viral particle. Second,
minimizing the
S homologous sequence between the trans-enzyme plasmid and packaging construct
will
reduce the probability of regenerating gag-pol through homologous
recombination. To do
this, one can modify the Vpr-RT-IN plasmid at the genetic level in at least
one of the
following ways:
(a). Eliminating incorporation of mRNA of Vpr-RT IN into vector particles.
Although there appears to be no lower limit to the size of virion-incorporated
mRNA in
virus particles, there does appear to be an upper limit. The genome of a
typical
replication-competent murine retrovirus is about 8.3 kb, whereas that of Rous
sarcoma
virus, which contains src sequences in addition to the normal complement of
viral genes,
is about 9.3 kb. The maximum size for a replication-competent spleen necrosis
virus
vector is similar, about 10 kb (Gelinas et al PNAS (1986)). In the case of HIV-
1, the size
of the virus genome is about 9.2 kb. It is believed that a size greater than
18 kb may
eliminate any possibility of packaging into the virus particle, particularly
without an
mRNA packaging signal. To do this, one may construct the Vpr-RT-IN expression
plasmid to also contain 1-18 kb of stuffer DNA, e.g., derived from lambda DNA
("Vpr-
RT-IN/ST"). These plasmids transcribe greater than 4 kb of mRNAs to express
the Vpr-
RT-IN fusion protein. To identify which of the Vpr-RT IN/st plasmids still
efficiently
provide RT and IN function in trans, one can test the different constructs
compared with
the parent plasmid, Vpr-RT-IN. (Fig. 27a)
(b). Eliminating incorporation of mRNA of packaging construct into vector
particles One can construct the Gag-PR or Gag-PR-RT-IN expression plasmids
containing
1-18 kb of stuffer DNA as described for (a), generating Gag-Pro/ST or Gag-PR-
RT-
IN/ST. These plasmids will transcribe greater than 7kb mRNA to express the
Gag/Gag-PR
or Gag/Gag-PR-RT-IN. To identify which of the Gag-Pro/ST or Gag-PR-RT-IN/ST
still
efficiently produce the high level of p24 antigen in the cell, one can test
the different sized
constructs against the parent plasmids, Gag-Pro or Gag-PR-RT-IN.
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(c). Humanized Vpr-RT INgene to reduce homologous sequence
Expression of HIV structural proteins and enzymes, like those of the gag,
reverse
transcriptase, integrase, and envelope protein, is facilitated by a cis-acting
element in the
viral mRNA known as the rev-responsive element (RRE), and by the action of Rev
since
these viral mRNA contain the multiple inhibitory sequences (INS) (Malim et at
(1989)
Nature 338:254-257; Brighty et al (1994) PNAS 91:8314-8313).
A current hypothesis about the function of INS is that they are binding sites
for
cellular factors that contribute to instability. Recently, it has been
demonstrated that
substitutions of AT-rich regions in gag and env, mostly in the third-site
position, without
changing the amino acid sequence of the produced Gag and Env proteins, can
result in
efficient expression of Gag and Env in a Rev- and RRE-independent fashion
(Schwartz et
al. (1992) J. Virol. 66:7176-7182; Haas et al (1996) Current Biology 6:315-
324). Recent
studies suggested that the codon-usage effects are a major impediment to the
efficient
expression of HIV-1 genes, e.g., rev. Therefore, the creation of a synthetic
coding
sequence based on codons over-represented in highly expressed human genes
overcomes a
major limitation to the translational efficiency of HIV-1 Env. Moreover, this
synthetic
gene can result in efficient expression of Env protein in a Rev- and RRE-
independent
fashion (Haas et al (1996) Current Biology 6:315-324). Previous studies have
indicated
the profound bias in the codon usage of the Pol proteins (RT and IN) (Kypr et
al (1987)
Nature 327:20).
A synthetic Vpr-RT-IN gene based on optimal codon usage (humanized Vpr-RT-
IN gene) will provide several advantages over the current Vpr-RT-IN gene.
First, since
about one-third of the sequence will change, the humanized Vpr-RT-IN plasmid
should
dramatically reduce the homologous recombination with the RT and IN part of
packaging
constructs. Second, the humanized synthetic gene can result in efficient
expression of
Vpr-RT-IN protein in a Rev- and RRE-independent fashion. Therefore, RRE
sequence,
which exists in both the packaging constuction and transfer vector, can be
removed from
the trans-enzyme plasmid. Fig. 27c shows the codon usage of highly expressed
human
genes (Haas et al (1997) Current Biology 6:315-324). Fig. 27b shows how to
humanize
Vpr, RT and IN genes by PCR.
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Example 12: A Retrovirus-Based Vector That Enables Transduction
of Non-dividing Cells
Lentiviruses such as HIV-1 can infect non-proliferating cells owing to the
karyophilic properties of the lentiviral preintegration complex which governs
recognition
of the cell nuclear import machinery. Lentivirus-based vectors (HIV, FIV and
EIAV) can
transduce cell lines that are growth-arrested in culture, as well as
macrophage, neuron,
muscle and retina. However, the greatest concern about the clinical use of HIV-
based
vectors is linked to the possibility that the parental pathogenic virus might
be reconstituted
through genetic recombination.
Until now, retroviral vectors used in clinical trials have been primarily
derived
from oncoretroviruses such as the murine leukemia virus (MLV). These vectors
can only
transduce cells that divide shortly after infection because the MLV
preintegration complex
cannot migrate to the nucleus in the absence of mitosis. This considerably
limits the use
of MLV-based vectors for gene delivery into targets such as neurons,
hepatocytes,
myocytes and hematopoietic stem cells.
In this example, we disclose a novel approach that splits the structure of
Gag/Gag-
PR-RT-IN into two separate coding sequences: one that express the Gag and
another that
express the Gag-PR-RT-IN (Fig. 28). The production of the fixed ratio of Gag
to Gag-PR-
RT-IN will be regulated by the IRES, a cis-acting element. Since the Gag and
Gag-PR-RT
IN express from the two separate coding sequences, and Gag-PR-RT-IN is not
required for
the conserved stem-loop structure and downstream mRNA secondary structure, one
can
insert specific sequences into the C-terminus region of gag, via linkage to
the nucleocapsid
protein (NC) (Fig. 28). Since NC associates with the PIC, the modified NC
containing a
nuclear localization signal or other specific motif that facilitates nuclear
transport will
enable the PIC of an MLV-based vector to be transported into the nucleus in
the absence
of mitosis. Therefore, the modified MLV based vector packaging system will
facilitate
MLV-based vectors to transduce nondividing cells.
Recent studies suggested that the central DNA flap acts as a cis-determinant
of
HIV-1 DNA nuclear import. The location of the central flap has been precisely
defined in
the case of HIV-1. Central strand displacement starts at the first nucleotide
following the
cPPT sequence and stops 99 nucleotides downstream at the terminator 2 site of
CTS. The
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presence of a DNA flap at the center of the genome can probably be generalized
to all
lentiviruses. Thus, the reverse-transcribed HIV-1 genome bears a cis-acting
determinant
for its nuclear import. HIV-1 gene transfer vectors lacking the central DNA
flap exhibit a
strong nuclear import defect. This defect can be rescued by reinsertion of the
DNA flap
sequence into the transfer vector.
Thus provided is an improved MLV-based transfer vector that contains a DNA
flat
sequence derived from HIV-1. To facilitate initiating the central strand
synthesis, the PPT
sequence derived from MLV is inserted upstream of the cPPT sequence (Fig. 29).
This
novel design of the MLV-based vector improves the ability to transduce non-
dividing
cells.
***
All patents and publications mentioned in the specification are indicative of
the
levels of skill of those skilled in the art to which the invention pertains.
All references
cited in this disclosure are incorporated by reference to the same extent as
if each
1 S reference had been incorporated by reference in its entirety individually.
One skilled in
the art would readily appreciate that the present invention is well adapted to
carry out the
objects and obtain the ends and advantages mentioned, as well as those
inherent. The
methods and systsems described herein are exemplary and not intended as
limitations on
the scope of the invention. It will be readily apparent to one skilled in the
art that varying
substitutions and modifications may be made to the invention disclosed herein
without
departing from the scope and spirit of the invention.
The invention illustratively described herein suitably may be practiced in the
absence of any element or elements, limitation or limitations which is not
specifically
disclosed herein. Thus, for example, in each instance herein any of the terms
"comprising", "consisting essentially of and "consisting of" may be replaced
with either
of the other two terms. The terms and expressions which have been employed are
used as
terms of description and not of limitation, and there is no intention that in
the use of such
terms and expressions of excluding any equivalents of the features shown and
described or
portions thereof, but it is recognized that various modifications are possible
within the
scope of the invention claimed. Thus, it should be understood that although
the present
CA 02379207 2002-O1-08
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invention has been specifically disclosed by preferred embodiments, optional
features,
modification and variation of the concepts herein disclosed may be resorted to
by those
skilled in the art, and that such modifications and variations are considered
to be within
the scope of this invention as defined by the description and the appended
claims.
S In addition, where features or aspects of the invention are described in
terms of
Markush groups or other grouping of alternatives, those skilled in the art
will recognize
that the invention is also thereby described in terms of any individual member
or subgroup
of members of the Markush group or other group.
Thus, additional embodiments are within the scope of the invention and within
the
following claims:
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SEQUENCE LISTING
<110> John C. Kappes
Xiaoyun Wu
<120> MEASURING GENETIC RECOMBINATION IN HIV
<130> 030908.0008-US
<140> 60/143,015
<141> 1999-07-09
<160> 6
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 9181
<212> DNA
<213> Artificial Sequence
<220>
<223> Primers
<400> 1
ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 60
tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 120
gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca 180
gtggcgcccg aacagggacc tgaaagcgaa agggaaacca gaggagctct ctcgacgcag 240
gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc 300
aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa 360
gcgggggaga attagatcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 420
ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 480
gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 540
agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 600
atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 660
acaaaagtaa gaaaaaagca cagcaagcag cagctgacac aggacacagc aatcaggtca 720
gccaaaatta ccctatagtg cagaacatcc aggggcaaat ggtacatcag gccatatcac 780
ctagaacttt aaatgcatgg gtaaaagtag tagaagagaa ggctttcagc ccagaagtga 840
tacccatgtt ttcagcatta tcagaaggag ccaccccaca agatttaaac accatgctaa 900
acacagtggg gggacatcaa gcagccatgc aaatgttaaa agagaccatc aatgaggaag 960
ctgcagaatg ggatagagtg catccagtgc atgcagggcc tattgcacca ggccagatga 1020
gagaaccaag gggaagtgac atagcaggaa ctactagtac ccttcaggaa caaataggat 1080
ggatgacaaa taatccacct atcccagtag gagaaattta taaaagatgg ataatcctgg 1140
gattaaataa aatagtaaga atgtatagcc ctaccagcat tctggacata agacaaggac 1200
caaaggaacc ctttagagac tatgtagacc ggttctataa aactctaaga gccgagcaag 1260
cttcacagga ggtaaaaaat tggatgacag aaaccttgtt ggtccaaaat gcgaacccag 1320
attgtaagac tattttaaaa gcattgggac cagcggctac actagaagaa atgatgacag 1380
catgtcaggg agtaggagga cccggccata aggcaagagt tttggctgaa gcaatgagcc 1440
aagtaacaaa ttcagctacc ataatgatgc agagaggcaa ttttaggaac caaagaaaga 1500
ttgttaagtg tttcaattgt ggcaaagaag ggcacacagc cagaaattgc agggccccta 1560
ggaaaaaggg ctgttggaaa tgtggaaagg aaggacacca aatgaaagat tgtactgaga 1620
gacaggctaa ttttttaggg aagatctggc cttcctacaa gggaaggcca gggaattttc 1680
ttcagagcag accagagcca acagccccac cagaagagag cttcaggtct ggggtagaga 1740
caacaactcc ccctcagaag caggagccga tagacaagga actgtatcct ttaacttccc 1800
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2
tcaggtcact ctttggcaac gacccctcgt cacaataaag ataggggggc aactaaagga 1860
agctctatta gatacaggag cagatgatac agtattagaa gaaatgagtt tgccaggaag 1920
atggaaacca aaaatgatag ggggaattgg aggttttatc aaagtaagac agtatgatca 1980
gatactcata gaaatctgtg gacataaagc tataggtaca gtattagtag gacctacacc 2040
tgtcaacata attggaagaa atctgttgac tcagattggt tgcactttaa attttcccat 2100
tagccctatt gagactgtac cagtaaaatt aaagccagga atggatggcc caaaagttaa 2160
acaatggcca ttgacagaag aaaaaataaa agcattagta gaaatttgta cagagatgga 2220
aaaggaaggg aaaatttcaa aaattgggcc tgaaaatcca tacaatactc cagtatttgc 2280
cataaagaaa aaagacagta ctaaatggag aaaattagta gatttcagag aacttaataa 2340
gagaactcaa gacttctggg aagttcaatt aggaatacca catcccgcag ggttaaaaaa 2400
gaaaaaatca gtaacagtac tggatgtggg tgatgcatat ttttcagttc ccttagatga 2460
agacttcagg aagtatactg catttaccat acctagtata aacaatgaga caccagggat 2520
tagatatcag tacaatgtgc ttccacaggg atggaaagga tcaccagcaa tattccaaag 2580
tagcatgaca aaaatcttag agccttttag aaaacaaaat ccagacatag ttatctatca 2640
atacatggat gatttgtatg taggatctga cttagaaata gggcagcata .gaacaaaaat 2700
agaggagctg agacaacatc tgttgaggtg gggacttacc acaccagaca aaaaacatca 2760
gaaagaacct ccattccttt ggatgggtta tgaactccat cctgataaat ggacagtaca 2820
gcctatagtg ctgccagaaa aagacagctg gactgtcaat gacatacaga agttagtggg 2880
gaaattgaat tgggcaagtc agatttaccc agggattaaa gtaaggcaat tatgtaaact 2940
ccttagagga accaaagcac taacagaagt aataccacta acagaagaag cagagctaga 3000
actggcagaa aacagagaga ttctaaaaga accagtacat ggagtgtatt atgacccatc 3060
aaaagactta atagcagaaa tacagaagca ggggcaaggc caatggacat atcaaattta 3120
tcaagagcca tttaaaaatc tgaaaacagg aaaatatgca agaatgaggg gtgcccacac 3180
taatgatgta aaacaattaa cagaggcagt gcaaaaaata accacagaaa gcatagtaat 3240
atggggaaag actcctaaat ttaaactgcc catacaaaag gaaacatggg aaacatggtg 3300
gacagagtat tggcaagcca cctggattcc tgagtgggag tttgttaata cccctccctt 3360
agtgaaatta tggtaccagt tagagaaaga acccatagta ggagcagaaa ccttctatgt 3420
agatggggca gctaacaggg agactaaatt aggaaaagca ggatatgtta ctaatagagg 3480
aagacaaaaa gttgtcaccc taactgacac aacaaatcag aagactgagt tacaagcaat 3540
ttatctagct ttgcaggatt cgggattaga agtaaacata gtaacagact cacaatatgc 3600
attaggaatc attcaagcac aaccagatca aagtgaatca gagttagtca atcaaataat 3660
agagcagtta ataaaaaagg aaaaggtcta tctggcatgg gtaccagcac acaaaggaat 3720
tggaggaaat gaacaagtag ataaattagt cagtgctgga atcaggaaag tactattttt 3780
agatggaata gataaggccc aagatgaaca tgagaaatat cacagtaatt ggagagcaat 3840
ggctagtgat tttaacctgc cacctgtagt agcaaaagaa atagtagcca gctgtgataa 3900
atgtcagcta aaaggagaag ccatgcatgg acaagtagac tgtagtccag gaatatggca 3960
actagattgt acacatttag aaggaaaagt tatcctggta gcagttcatg tagccagtgg 4020
atatatagaa gcagaagtta ttccagcaga aacagggcag gaaacagcat attttctttt 4080
aaaattagca ggaagatggc cagtaaaaac aatacatact gacaatggca gcaatttcac 4140
cggtgctacg gttagggccg cctgttggtg ggcgggaatc aagcaggaat ttggaattcc 4200
ctacaatccc caaagtcaag gagtagtaga atctatgaat aaagaattaa agaaaattat 4260
aggacaggta agagatcagg ctgaacatct taagacagca gtacaaatgg cagtattcat 4320
ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga 4380
cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa 4440
ttttcgggtt tattacaggg acagcagaaa tccactttgg aaaggaccag caaagctcct 4500
ctggaaaggt gaaggggcag tagtaataca agataatagt gacataaaag tagtgccaag 4560
aagaaaagca aagatcatta gggattatgg aaaacagatg gcaggtgatg attgtgtggc 4620
aagtagacag gatgaggatt agaacatgga aaagtttagt aaaacaccat atgtatgttt 4680
cagggaaagc taggggatgg ttttatagac atcactatga aagccctcat ccaagaataa 4740
gttcagaagt acacatccca ctaggggatg ctagattggt aataacaaca tattggggtc 4800
tgcatacagg agaaagagac tggcatttgg gtcagggagt ctccatagaa tggaggaaaa 4860
agagatatag cacacaagta gaccctgaac tagcagacca actaattcat ctgtattact 4920
ttgactgttt ttcagactct gctataagaa aggccttatt aggacacata gttagcccta 4980
ggtgtgaata tcaagcagga cataacaagg taggatctct acaatacttg gcactagcag 5040
cattaataac accaaaaaag ataaagccac ctttgcctag tgttacgaaa ctgacagagg 5100
atagatggaa caagccccag aagaccaagg gccacagagg gagccacaca atgaatggac 5160
actagagctt ttagaggagc ttaagaatga agctgttaga cattttccta ggatttggct 5220
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3
ccatggctta gggcaacata tctatgaaac ttatggggat acttgggcag gagtggaagc 5280
cataataaga attctgcaac aactgctgtt tatccatttt cagaattggg tgtcgacata 5340
gcagaatagg cgttactcga cagaggagag caagaaatgg agccagtaga tcctagacta 5400
gagccctgga agcatccagg aagtcagcct aaaactgctt gtaccaattg ctattgtaaa 5460
aagtgttgct ttcattgcca agtttgtttc ataacaaaag ccttaggcat ctcctatggc 5520
aggaagaagc ggagacagcg acgaagagct catcagaaca gtcagactca tcaagcttct 5580
ctatcaaagc agtaagtagt acatgtaatg caacctatac caatagtagc aatagtagca 5640
ttagtagtag caataataat agcaatagtt gtgtggtcca tagtaatcat agaatatagg 5700
aaaatattaa gacaaagaaa aatagacagg ttaattgata gactaataga aagagcagaa 5760
gacagtggca atgagagtga aggagaaata tcagcacttg tggagatggg ggtggagatg 5820
gggcaccatg ctccttggga tgttgatgat ctgtagtgct acagaaaaat tgtgggtcac 5880
agtctattat ggggtacctg tgtggaagga agcaaccacc actctatttt gtgcatcaga 5940
tgctaaagca tatgatacag aggtacataa tgtttgggcc acacatgcct gtgtacccac 6000
agaccccaac ccacaagaag tagtattggt aaatgtgaca gaaaatttta acatgtggaa 6060
aaatgacatg gtagaacaga tgcatgagga tataatcagt ttatgggatc aaagcctaaa 6120
gccatgtgta aaattaaccc cactctgtgt tagtttaaag tgcactgatt tgaagaatga 6180
tactaatacc aatagtagta gcgggagaat gataatggag aaaggagaga taaaaaactg 6240
ctctttcaat atcagcacaa gcataagagg taaggtgcag aaagaatatg cattttttta 6300
taaacttgat ataataccaa tagataatga tactaccagc tataagttga caagttgtaa 6360
cacctcagtc attacacagg cctgtccaaa ggtatccttt gagccaattc ccatacatta 6420
ttgtgccccg gctggttttg cgattctaaa atgtaataat aagacgttca atggaacagg 6480
accatgtaca aatgtcagca cagtacaatg tacacatgga attaggccag tagtatcaac 6540
tcaactgctg ttaaatggca gtctagcaga agaagaggta gtaattagat ctgtcaattt 6600
cacggacaat gctaaaacca taatagtaca gctgaacaca tctgtagaaa ttaattgtac 6660
aagacccaac aacaatacaa gaaaaagaat ccgtatccag agaggaccag ggagagcatt 6720
tgttacaata ggaaaaatag gaaatatgag acaagcacat tgtaacatta gtagagcaaa 6780
atggaataac actttaaaac agatagctag caaattaaga gaacaatttg gaaataataa 6840
aacaataatc tttaagcaat cctcaggagg ggacccagaa attgtaacgc acagttttaa 6900
ttgtggaggg gaatttttct actgtaattc aacacaactg tttaatagta cttggtttaa 6960
tagtacttgg agtactgaag ggtcaaataa cactgaagga agtgacacaa tcaccctccc 7020
atgcagaata aaacaaatta taaacatgtg gcagaaagta ggaaaagcaa tgtatgcccc 7080
tcccatcagt ggacaaatta gatgttcatc aaatattaca gggctgctat taacaagaga 7140
tggtggtaat agcaacaatg agtccgagat cttcagacct ggaggaggag atatgaggga 7200
caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 7260
acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 7320
tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcct caatgacgct 7380
gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 7440
ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 7500
ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 7560
ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 7620
atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 7680
ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 7740
acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 7800
ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 7860
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 7920
tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 7980
tggagagaga gacagagaca gatccattcg attagtgaac ggatccttgg cacttatctg 8040
ggacgatctg cggagcctgt gcctcttcag ctaccaccgc ttgagagact tactcttgat 8100
tgtaacgagg attgtggaac ttctgggacg cagggggtgg gaagccctca aatattggtg 8160
gaatctccta cagtattgga gtcaggaact aaagaatagt gctgttagct tgctcaatgc 8220
cacagccata gcagtagctg aggggacaga tagggttata gaagtagtac aaggagcttg 8280
tagagctatt cgccacatac ctagaagaat aagacagggc ttggaaagga ttttgctata 8340
agatgggtgg caagtggtca aaaagtagtg tgattggatg gcctactgta agggaaagaa 8400
tgagacgagc tgagccagca gcagataggg tgggagcagc atctcgagac ctggaaaaac 8460
atggagcaat cacaagtagc aatacagcag ctaccaatgc tgcttgtgcc tggctagaag 8520
cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga 8580
cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc 8640
CA 02379207 2002-O1-08
WO 01/04360 PCT/US00/1859'7
4
taattcactc ccaaagaaga caagatatcc ttgatctgtg gatctaccac acacaaggct 8700
acttccctga ttagcagaac tacacaccag ggccaggggt cagatatcca ctgacctttg 8760
gatggtgcta caagctagta ccagttgagc cagataagat agaagaggcc aataaaggag 8820
agaacaccag cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag 8880
tgttagagtg gaggtttgac agccgcctag catttcatca cgtggcccga gagctgcatc 8940
cggagtactt caagaactgc tgacatcgag cttgctacaa gggactttcc gctggggact 9000
ttccagggag gcgtggcctg ggcgggactg gggagtggcg agccctcaga tcctgcatat 9060
aagcagctgc tttttgcctg tactgggtct ctctggttag accagatctg agcctgggag 9120
ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt 9180
c 9181
<210> 2
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primers
<400> 2
ccggaattct ggctaactag ggaacccact gc 32
<210> 3
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Primers
<400> 3
cgcggatcct tattgtgacg aggggtcgct gcc 33
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primers
<400> 4
gacctccata gaagacaccg 20
<210> 5
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Primers
<400> 5
ccggaattca tggagccagt agatcctaga c 31
<210> 6
<211> 31
<212> DNA
cacggacaat gctaaaacca taatagtaca gctgaacaca tct
CA 02379207 2002-O1-08
WO 01/04360 PCT/tTS00/18597
<213> Artificial Sequence
<220>
<223> Primers
<400> 6
cgcggatccg cagtgggttc cctagttagc c 31
