Note: Descriptions are shown in the official language in which they were submitted.
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
S "SHUFFLING OF CODON ALTERED GENES," Attorney Docket No. 02-028500, by
Patten and Stemmer, filed 09/29/98, and 60/117,729, "SHUFFLING OF CODON
ALTERED
GENES," Attorney Docket No. 02-028510, by Patten and Stemmer, filed January
29, 1999.
The application is also related to USSN 60/118,813 "OLIGONUCLEOTIDE MEDIATED
NUCLEIC ACID RECOMBINATION," by Crameri et al., Attorney Docket Number 02-296,
filed February 5, 1999; and USSN 60/141,049 "OLIGONUCLEOTIDE MEDIATED
NUCLEIC ACID RECOMBINATION," by Crameri et al., Attorney Docket Number 02-296-
1, filed June 24, 1999.
BACKGROUND
The genetic code is highly degenerate. Every DNA/RNA triplet (codon)
encoding an amino acid can typically be altered, with the exception of ATG/AUG
(coding for
methionine) and TGG/LTGG (coding for Tryptophan), without altering the
sequence of the
protein encoded by the corresponding nucleic acid sequence. Roughly, on
average (the
distribution of amino acids varies from protein to protein), each coding
triplet can be
substituted about 3 different ways, since there are 61 codons encoding 20
amino acids (there
are 3 additional triplets encoding stop codons, for a total of 64 codons
encoding 20 amino
acids). This represents a possible sequence diversity of approximately 3"
possible sequences
which encode a given protein, where n is the length of the protein in amino
acids. As can
easily be seen, for proteins of even modest length, the number of possible
nucleic acids which
can encode the protein exceeds the number of physical particles in the
universe (estimated at
about 10$° particles).
This tremendous potential coding sequence space for individual proteins has
interesting evolutionary implications. For example, hypermutable viruses such
as HIVs and
other retroviruses typically stay one step ahead of the host immune system by
accumulating
non-random mutations based, in part, upon the particular codons used to encode
recognition
molecules, e.g., in the envelope portion of the virus. The mutations are non-
random because
viruses are selected for the ability to mutate to forms which are not quickly
recognized by the
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
host immune system. A consequence of this is that viruses are selected to have
a non-random
set of codons encoding, e.g., envelope proteins, allowing the viruses to shift
forms rapidly by
making, e.g., specific point mutations to generate specific alterations in
protein structure.
Codon use is also non-random within species. By preferentially making a
subset of all possible t-RNAs, cells may conserve energy, and can optimize, ar
even regulate,
the efficiency of cellular translation systems. This fact has long been
recognized empirically,
often allowing investigators initially to determine the reading frame of a
given nucleic acid
sequence simply by consideration of the codons resulting from different
potential reading
frames. One consequence of this "species codon bias" is that proteins within a
species have a
limited set of possible mutations that can arise as a consequence of, e.g.,
point mutation. This
limits the possible evolution rate of proteins.
In addition to the diversity of nucleic acid coding sequences which encode any
given protein, it is now clear that protein sequences are, themselves, quite
degenerate. Often,
many of the amino acid residues constituting a protein may be substituted for
structurally
I 5 similar amino acid units without significantly changing the tertiary
structure of the protein.
Thus, it may be difficult to determine which residues to modify or to improve
desirable
properties of a protein.
For proteins which are commercially valuable, it would be desirable to be able
to gain access to a mutational spectrum which is different than that of the
native protein. The
present invention provides this, and many other features, that will be
apparent upon complete
review of the following.
SUMMARY OF THE INVENTION
The present invention provides methods of accessing a completely different
mutational spectrum for a selected protein than is available in the naturally
occurnng nucleic
acid encoding the protein. This increases the type and rate of forced
evolution for the
selected protein, allowing for rapid improvement of any detectable
characteristic of the
protein. In the methods, nucleic acids are synthesized with altered codon
usage, and/or
which encode one or several amino acid residue changes as compared to the
selected protein,
where the amino acid and codon usage changes can be conservative or non-
conservative.
The resulting codon/amino acid modified nucleic acids) are recombined using
DNA
shuffling techniques with either the native nucleic acid, or with each other
(or both), typically
2
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
using recursive shuffling methods. The nucleic acids or the encoded protein
are then
screened for a desirable property.
Thus, the invention provides methods of making colon altered nucleic acids.
In the methods, a first nucleic acid sequence encoding a first polypeptide
sequence is
selected. A plurality of colon altered nucleic acid sequences, each of which
encode the first
polypeptide, or a modified form thereof, are then selected (e.g., a library of
colon altered
nucleic acids can be selected in a biological assay which recognizes library
components or
activities), and the plurality of colon altered nucleic acid sequences is
recombined to produce
a target colon altered nucleic acid encoding a second protein. The target
colon altered
nucleic acid is then screened for a detectable functional or structural
property, optionally
including comparison to the properties of the first polypeptide. The goal of
such screening is
to identify a polypeptide that has a structural or functional property
equivalent or superior to
the first polypeptide. A nucleic acid encoding such a polypeptide can be used
in essentially
any procedure desired, including introducing the target colon altered nucleic
acid into a cell,
vector, virus, attenuated virus (e.g., as a component of a vaccine or
immunogenic
composition), transgenic organism, or the like.
Kits and compositions for practicing the methods are also provided, including
one or more of cell recombination mixtures and substrates (e.g., nucleic acids
with altered
colon usage), containers, instructional material for practicing the methods,
or the like.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a nucleic acid/amino acid sequence of a part of the monkey EPO
gene, which is similar to the human EPO gene.
Figure 2 shows an example of a colon altered EPO nucleic acid sequence.
Figure 3 shows an alignment of naturally occurring EPOS.
Figure 4 is a schematic of the human EPO wobble sequence space.
Figure 5 is a schematic of Mammalian EPO Family-Wobble Sequence Space.
Figure 6 is a sequence alignment of G-CSF homologs, with species
information.
Figure 7 is a sequence alignment of G-CSF homologs, with differences broken
out.
3
CA 02331335 2000-12-21
WO 00/18906 PCTNS99/22588
Figure 8 is a sequence alignment showing the hydrophobic core residues of
human G-CSF (blacked out).
Figure 9 is a schematic showing the shuffling strategy for G-CSF.
Figure 10 is a list of oligos used to make a codon altered alkaline
phosphatase.
Figure 11 is a map of oligos used to make a codon altered alkaline
phosphatase.
Figure 12 is a schematic of vaccination with evolution defective viruses.
Figure 13 is a schematic of different mutations that result from different
codon
types for ser, arg, and leu.
Figure 14 is a schematic of vaccination with evolution defective viruses.
Figure 15 is a schematic of vaccination with evolution defective viruses
showing sophisticated versus non-sophisticated "mutant clouds."
Figure 16, panels A-C show results of single mutations of different codons for
ser, arg, and leu.
Fig. 17 is a schematic of protein evolution with expanded mutation spectra.
Fig. 18, panels A-D show codon altered forms of Env.
Fig. 19 is a list of oligos in one application for synthesis of HIV Env.
DEFINITIONS
Unless clearly indicated to the contrary, the following definitions supplement
definitions of terms known in the art.
As used herein, a "recombinant" nucleic acid is a nucleic acid produced by
recombination between two or more nucleic acids, or any nucleic acid made by
an in vitro or
artificial process. The term "recombinant" when used with reference to a cell
indicates that
the cell comprises (and optionally replicates) a heterologous nucleic acid, or
expresses a
peptide or protein encoded by a heterologous nucleic acid. Recombinant cells
can contain
genes that are not found within the native (non-recombinant) wild-type form of
the cell.
Recombinant cells can also contain genes found in the native form of the cell
where the genes
are modified and re-introduced into the cell by artificial means. The term
also encompasses
cells that contain a nucleic acid endogenous to the cell that has been
artificially modified
without removing the nucleic acid from the cell; such modifications include
those obtained
by gene replacement, site-specific mutation, chimeraplasty, and related
techniques.
4
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
A "codon altered" nucleic acid is a first nucleic acid that encodes a first
polypeptide similar or identical to a naturally occurring polypeptide encoded
by a naturally
occurring nucleic acid, where the first nucleic acid utilizes a plurality of
codons to encode the
first polypeptide, which differ from the codons of the naturally occurring
nucleic acid that
encode the naturally occurring polypeptide.
A "nucleic acid sequence" refers to either a nucleic acid (e.g., RNA, DNA or
modified form thereof, in isolated, recombinant or native form) or to a
representation of the
nucleic acid such as a sequence of letters indicating the primary structure
(sequence) of the
nucleic acid.
A "polypeptide sequence" refers to either a polypeptide (or modified form
thereof, in isolated, recombinant or native form) or to a representation of
the polypeptide
such as a sequence of letters or other character string information indicating
the primary
structure (amino acid sequence) of the polypeptide.
A "modified form" of a reference polypeptide is a target polypeptide which
has a similar, but not identical, sequence to the reference polypeptide. The
sequence of the
target polypeptide can differ from the reference polypeptide by conservative
or non-
conservative substitutions of the reference polypeptide sequence. As noted in
more detail,
supra, different nucleic acids encoding different target polypeptides having
different non-
conservative substitutions relative to the reference polypeptide can be
recombined to produce
a recombined nucleic acid encoding a target polypeptide more similar to the
reference
polypeptide.
A "plurality of forms" of a selected nucleic acid refers to a plurality of
homologs of the nucleic acid. The homologs can be from naturally occurring
homologs (e.g.,
two or more homologous genes, or derivatives thereof) or by artificial
synthesis of one or
more nucleic acids having related sequences, or by modification of one or more
nucleic acid
to produce related nucleic acids. Nucleic acids are homologous when they are
derived,
naturally or artificially, from a common ancestor sequence. During natural
evolution, this
occurs when two or more descendent sequences diverge from a parent sequence
over time,
i.e., due to mutation and natural selection. Under artificial conditions,
divergence occurs,
e.g., in one of two ways. First, a given sequence can be artificially
recombined with another
sequence, as occurs, e.g., during typical cloning, to produce a descendent
nucleic acid.
S
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
Alternatively, a nucleic acid can be synthesized de novo, by synthesizing a
nucleic acid
which varies in sequence from a given parental nucleic acid sequence.
When there is no explicit knowledge about the ancestry of two nucleic acids,
homology is typically inferred by sequence comparison between two sequences.
Where two
nucleic acid sequences show sequence similarity it is inferred that the two
nucleic acids share
a common ancestor. The precise level of sequence similarity required to
establish homology
varies in the art depending on a variety of factors. For purposes of this
disclosure, two
sequences are considered homologous where they share sufficient sequence
identity to allow
recombination to occur between two nucleic acid molecules, or when codon
changes can be
made which would result in two or more nucleic acids having the ability to
recombine.
Typically, nucleic acids require regions of close similarity spaced roughly
the same distance
apart to permit recombination to occur.
The terms "identical" or percent "identity," in the context of two or more
nucleic acid or polypeptide sequences, refer to two or more sequences or
subsequences that
are the same or have a specified percentage of amino acid residues or
nucleotides that are the
same, when compared and aligned for maximum correspondence, as measured using
one of
the sequence comparison algorithms described below (or other algorithms
available to
persons of skill) or by visual inspection.
The phrase "substantially identical," in the context of two nucleic acids or
polypeptides refers to two or more sequences or subsequences that have at
least about 40%,
50%, 60%, or preferably about 70% or 80% or more, or most preferably 90-95%
nucleotide
or amino acid residue identity, when compared and aligned for maximum
correspondence, as
measured using one of the following sequence comparison algorithms or by
visual inspection.
Such "substantially identical" sequences are typically considered to be
homologous.
Preferably, the "substantial identity" exists over a region of the sequences
that is at least
about 50 residues in length, more preferably over a region of at least about
100 residues, and
most preferably the sequences are substantially identical over at least about
150 residues, or
over the full length of the two sequences to be compared.
For sequence comparison and homology determination, typically one
sequence acts as a reference sequence to which test sequences are compared.
When using a
sequence comparison algorithm, test and reference sequences are input into a
computer,
6
CA 02331335 2000-12-21
WO 00/18906 PCTNS99/22588
subsequence coordinates are designated, if necessary, and sequence algorithm
program
parameters are designated. The sequence comparison algorithm then calculates
the percent
sequence identity for the test sequences) relative to the reference sequence,
based on the
designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the
local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 ( I981 ),
by the
homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (
1970), by the
search for similarity method of Pearsan & Lipman, Proc. Nat'l. Acad. Sci. USA
85:2444
(1988), by computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group,
575
Science Dr., Madison, WI), or by visual inspection (see generally, Ausubel et
al., infra).
One example of algorithm that is suitable for determining percent sequence
identity and sequence similarity is the BLAST algorithm, which is described in
Altschul et
al., J. Mol. Biol. 215:403-410 ( 1990). Software for performing BLAST analyses
is publicly
available through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high
scoring
sequence pairs (HSPs) by identifying short words of length W in the query
sequence, which
either match or satisfy some positive-valued threshold score T when aligned
with a word of
the same length in a database sequence. T is referred to as the neighborhood
word score
threshold (Altschul et al., supra). These initial neighborhood word hits act
as seeds for
initiating searches to find longer HSPs containing them. The word hits are
then extended in
both directions along each sequence for as far as the cumulative alignment
score can be
increased. Cumulative scores are calculated using, for nucleotide sequences,
the parameters
M (reward score for a pair of matching residues; always > 0) and N (penalty
score for
mismatching residues; always < 0). For amino acid sequences, a scoring matrix
is used to
calculate the cumulative score. Extension of the word hits in each direction
are halted when:
the cumulative alignment score falls off by the quantity X from its maximum
achieved value;
the cumulative score goes to zero or below, due to the accumulation of one or
more negative-
scoring residue alignments; or the end of either sequence is reached. The
BLAST algorithm
parameters W, T, and X determine the sensitivity and speed of the alignment.
The BLASTN
program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation
7
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
(E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For
amino acid
sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an
expectation (E)
of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff ( 1989) Proc.
Natl.
Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm
also performs a statistical analysis of the similarity between two sequences
(see, e.g., Karlin
& Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of
similarity
provided by the BLAST algorithm is the smallest sum probability (P(N)), which
provides an
indication of the probability by which a match between two nucleotide or amino
acid
sequences would occur by chance. For example, a nucleic acid is considered
similar to a
reference sequence if the smallest sum probability in a comparison of the test
nucleic acid to
the reference nucleic acid is less than about 0.1, more preferably less than
about 0.01, and
most preferably less than about 0.001.
Another indication that two nucleic acid sequences are substantially
identical/
homologous is that the two molecules hybridize to each other under stringent
conditions.
The phrase "hybridizing specifically to," refers to the binding, duplexing, or
hybridizing of a
molecule only to a particular nucleotide sequence under stringent conditions,
including when
that sequence is present in a complex mixture (e.g., total cellular) DNA or
RNA. "Bind(s)
substantially" refers to complementary hybridization between a probe nucleic
acid and a
target nucleic acid and embraces minor mismatches that can be accommodated by
reducing
the stringency of the hybridization media to achieve the desired detection of
the target
polynucleotide sequence.
"Stringent hybridization conditions" and "stringent hybridization wash
conditions" in the context of nucleic acid hybridization experiments such as
Southern and
northern hybridizations are sequence dependent, and are different under
different
environmental parameters. Longer sequences and sequences with higher G:C
content remain
hybridized at higher temperatures (or at lower sait). An extensive guide to
the hybridization
of nucleic acids is found in Tijssen ( 1993) Laboratory Techniques in
Biochemistry and
Molecular Biology--Hybridization with Nucleic Acid Probes part I chapter 2
"Overview of
principles of hybridization and the strategy of nucleic acid probe assays,"
Elsevier, New
York.
CA 02331335 2000-12-21
WO 00/18906 PCT/IJS99/22588
Generally, highly stringent hybridization and wash conditions are selected to
be about 5 °C lower than the thermal melting point (Tm) for the
specific sequence at a defined
ionic strength and pH. Typically, under "stringent conditions" a probe will
hybridize to its
target subsequence, but not to unrelated (non-homologous) sequences.
The Tm is the temperature (under defined ionic strength and pH) at which 50%
of the target sequence hybridizes to a perfectly matched probe. Very stringent
conditions are
selected to be equal to the Tm for a particular probe. An example of stringent
hybridization
conditions for hybridization of complementary nucleic acids which have more
than 100
complementary residues on a filter in a Southern or northern blot is 50%
formamide with 1
mg of heparin at 42 °C, with the hybridization being carried out
overnight. An example of
highly stringent wash conditions is O.15M NaCI at 72 °C for about 15
minutes. An example
of stringent wash conditions is a 0.2x SSC wash at 65 °C for 15 minutes
(see, Sambrook,
infra., for a description of SSC buffer). Often, a high stringency wash is
preceded by a low
stringency wash to remove background probe signal. An example medium
stringency wash
for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45 °C
for 15 minutes. An
example low stringency wash for a duplex of, e.g., more than 100 nucleotides,
is 4-6x SSC at
40 °C for 15 minutes. For short probes (e.g., about 10 to 50
nucleotides), stringent conditions
typically involve salt concentrations of less than about 1.0 M Na ion,
typically about 0.01 to
i .0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the
temperature is typically
at least about 40 °C. Stringent conditions can also be achieved with
the addition of
destabilizing agents such as formamide. In general, a signal to noise ratio of
2x (or higher)
than that observed for an unrelated probe in the particular hybridization
assay indicates
detection of a specific hybridization. If the signal to noise ratio is less
than 2x binding of an
unrelated probe (e.g., a nucleic acid encoding a non-homologous protein), the
nucleic acids at
issue do not hybridize under stringent conditions. Similarly, if the signal to
noise ratio is less
than 25% as high as that observed for a perfectly matched probe under
stringent conditions,
the nucleic acids do not "hybridize under stringent conditions" as that term
is used herein.
This does not apply to highly stringent conditions, as the stringency can
theoretically be
increased until only a perfectly matched probe will hybridize.
In one example hybridization procedure, a target nucleic acid to be probed is
blotted onto a filter by any conventional method. An unrelated nucleic acid
such as a plasmid
9
CA 02331335 2000-12-21
WO 00/18906 PCTNS99/22588
vector (assuming that the target nucleic acid has no homology with the target
nucleic acid) is
also blotted, in approximately equal amounts onto the filter. The filter is
probed with a
labeled probe complementary to the target nucleic acid. The experiment is
repeated at
gradually increasing stringency of hybridization and wash conditions until
signal from the
hybridization of the labeled probe to the complementary target is 10-100X as
high as to the
unrelated plasmid vector nucleic acid. Once these conditions are determined as
described
above, a test nucleic acid is probed under the same conditions as the target.
If signal from the
labeled probe is 25% as high or higher than the signal from binding of the
probe to the target,
the test nucleic acid "hybridizes under stringent conditions" to the probe. If
the signal is less
than 25% as high, the test nucleic acid does not hybridize under stringent
conditions to the
probe.
Nucleic acids which do not hybridize to each other under stringent conditions
are still recognizable as variant forms of a nucleic acid when the
polypeptides they encode
are substantially identical. This occurs, e.g., when a copy of a nucleic acid
is created using
the maximum codon degeneracy permitted by the genetic code. Such nucleic acids
are not
functionally equivalent, as described in detail herein, due to differences in
mRNA folding,
alterations of regulatory sequences and the like.
Another indication that two nucleic acid sequences or polypeptides are variant
forms is that the polypeptide encoded by the first nucleic acid is
immunologically cross
reactive with the polypeptide encoded by the second nucleic acid, as tested by
polyclonal
antisera generated to the first polypeptide. Thus, a poiypeptide is typically
substantially
identical to a second polypeptide, for example, where the two peptides differ
only by
conservative substitutions.
"Conservatively modified variations" of a particular polynucleotide sequence
are those polynucleotide variations that encode identical or essentially
identical amino acid
sequences, or where the polynucleotide does not encode an amino acid sequence,
which
encode essentially identical sequences. Because of the degeneracy of the
genetic code, a large
number of functionally identical nucleic acids encode any given polypeptide.
For instance,
the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid
arginine.
Thus, at every position where an arginine is specified by a codon, the codon
can be altered to
any of the corresponding codons described without altering the encoded
polypeptide. Such
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
nucleic acid variations are "silent variations," which are one species of
"conservatively
modified variations." Every polynucleotide sequence described herein which
encodes a
polypeptide also optionally describes every possible silent variation, except
where otherwise
noted. One of skill will recognize that each codon in a nucleic acid (except
AUG, which is
ordinarily the codon for methionine, and TGG, which is ordinarily the codon
for tryptophan)
can be modified to yield a peptide which is structurally identical.
Furthermore, one of skill will recognize that individual substitutions,
deletions
or additions which alter, add or delete a single amino acid or a small
percentage of amino
acids (typically less than S%, more typically less than 1 %) in an encoded
sequence are
"conservatively modified variations" where the alterations result in the
substitution of an
amino acid with a chemically similar amino acid. Conservative substitution
tables providing
functionally similar amino acids are well known in the art. The following five
groups each
contain amino acids that are conservative substitutions for one another:
Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I);
Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing:
Methionine
(M), Cysteine (C);Basic: Arginine (R), Lysine (K), Histidine (H); Acidic:
Aspartic acid (D),
Glutamic acid (E), Asparagine (N), Glutamine (Q). See also, Creighton (1984)
Proteins,
W.H. Freeman and Company. In addition, individual substitutions, deletions or
additions
which alter, add or delete a single amino acid or a small percentage of amino
acids in an
encoded sequence are also "conservatively modified variations." Sequences that
differ by
conservative variations are generally homologous.
The term "isolated", when applied to a nucleic acid or protein, denotes that
the
nucleic acid or protein is essentially free of other cellular or other
components (e.g., library
components) with which it is associated in the natural state.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the
term encompasses nucleic acids containing known analogues of natural
nucleotides which
have similar binding properties as the reference nucleic acid and are
metabolized in a manner
similar to naturally occurring nucleotides. Unless otherwise indicated, a
particular nucleic
acid sequence also implicitly encompasses conservatively modified variants
thereof (e.g.
degenerate codon substitutions) and complementary sequences and as well as the
sequence
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
explicitly indicated. Specifically, degenerate codon substitutions may be
achieved by
generating sequences in which the third position of one or more selected (or
all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et al. ( 1991
) Nucleic Acid
Res. 19: 5081; Ohtsuka et al. ( 1985) J. Biol. Chem. 260: 2605-2608; Cassol et
al. ( 1992) ;
Rossolini et al. (1994) Mol. Cell. Probes 8: 91-98). The term nucleic acid is
generic to the
terms "gene", "DNA," "cDNA", "oligonucleotide," "RNA," "mRNA," and the like.
"Nucleic acid derived from a gene" refers to a nucleic acid for whose
synthesis the gene, or a subsequence thereof, has ultimately served as a
template. Thus, an
mRNA, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that
cDNA, a
DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc.,
are all
derived from the gene and detection of such derived products is indicative of
the presence
and/or abundance of the original gene and/or gene transcript in a sample.
A nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid sequence. For instance, a promoter or
enhancer is
operably linked to a coding sequence if it increases the transcription of the
coding sequence.
A "recombinant expression cassette" or simply an "expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically, with nucleic
acid elements
that are capable of effecting expression of a structural gene in hosts
compatible with such
sequences. Expression cassettes include at least promoters and optionally,
transcription
termination signals. Typically, the recombinant expression cassette includes a
nucleic acid to
be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a
promoter.
Additional factors necessary or helpful in effecting expression may also be
used as described
herein. For example, an expression cassette can also include nucleotide
sequences that
encode a signal sequence that directs secretion of an expressed protein from
the host cell.
Transcription termination signals, enhancers, and other nucleic acid sequences
that influence
gene expression, can also be included in an expression cassette.
DETAILED DISCUSSION OF THE INVENTION
In the present invention, the sequence diversity of substrates for DNA
shuffling procedures is increased by using codon-altered nucleic acids as
templates and/or by
using templates that encode proteins with conservative or non-conservative
amino acid
modifications as compared to a selected wild-type protein.
12
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
These codon altered nucleic acids can be chemically synthesized (e.g., using
standard artificial synthetic protocols, e.g., those typically used by
commercial sources from
which nucleic acids can be ordered), or can be made using any of a variety of
methods herein
of available to one of skill. For example, oligonucleotide fragments can be
made which
correspond to a codon altered nucleic acid which is desired using standard
synthetic methods,
followed by polymerase and/or ligase mediated oligonucleotide
ligation/recombination
protocols to generate full-length nucleic acids.
The combination of codon usage modifications and coding modifications can
be extensive enough to reduce or, under stringent conditions, even eliminate
the hybridization
of the codon-altered nucleic acids to a nucleic acid which naturally encodes
the selected
protein. This dramatically alters the mutations which result from possible
single nucleotide
mutations, providing access to greater diversity for DNA shuffling protocols.
In addition, the recombination and selection of such nucleic acids during DNA
shuffling procedures can result not only in access to a different set of
possible mutations, but
can also result in modified forms of transcriptional or translational
regulation, alterations in
nucleic acid localization, mRNA stability and the like. Furthermore, the
modified
hybridization properties of codon altered nucleic acids leads to alterations
in the ability of the
nucleic acids to hybridize with potential recombination partners, altering,
and ultimately
increasing, the available recombination diversity during shuffling.
Furthermore, "family shuffling" using codon-altered substrates even further
increases the possible sequence diversity of the starting materials for
recombination. As
currently practiced, family shuffling methods involve shuffling nucleic acids
encoding
sequence variants of a given protein (e.g., species or allele homologs). In
the present
methods, this procedure is modified by generating codon-altered versions of
the sequence
variants to access additional molecular diversity during recombination.
Additional diversity
is achieved by conservatively and non-conservatively modifying the starting
nucleic acids to
encode non-naturally occurring sequence variants. Family shuffling can be
performed even
using homologs of relatively low identity. In such cases, codons may be
changed in one or
more of the family members to increase the level of identity between the
members, thereby
increasing their ability to recombine using the methods of this invention.
13
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
Gene shuffling and family shuffling provide two of the most powerful
methods available for improving and "migrating" (gradually changing the type
of reaction,
substrate or activity of a selected protein such as an enzyme, or regulation
or structure of an
expressed component) the functions of proteins. In family shuffling,
homologous sequences,
e.g., from different species, chromosomal positions, or due to synthetic
alteration, are
recombined. In gene shuffling, a single sequence is mutated or otherwise
altered and then
recombined.
The generation and screening of high quality shuffled libraries provides for
DNA shuffling (or "directed evolution"). The availability of appropriate high-
throughput
analytical chemistry to screen the libraries permits integrated high-
throughput shuffling and
screening of the libraries to achieve a desired activity.
In one significant embodiment, oligonucleotides for constructing codon-
modified nucleic acids are designed in a computer ("in silico"). Predicted
colon-modified
recombinant nucleic acids can also be determined in silico, i.e., essentially
as taught in
Selifonov and Stemmer "METHODS FOR MAKING CHARACTER STRINGS,
POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS"
filed 02/05/1999, USSN 60/118854.
Furthermore, rather than generating colon-modified nucleic acids as
substrates for recombination, families of nucleic acids can be recombined
simply by
appropriate selection of the relevant oligonucleotides which are used in gene
reconstruction
methods to produce recombinant nucleic acids, i.e., by using colon-modified
nucleic acid
oligonucleotides as discussed herein in conjunction with family
oligonucleotide-mediated
shuffling methods, e.g., as taught in Crameri et al. "OLIGONUCLEOTIDE MEDIATED
NUCLEIC ACID RECOMBINATION" filed February 5, 1999, USSN 60/118,813 and
Crameri et al. "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION"
filed June 24, 1999, USSN 60/141,049. The technique can be used to recombine
homologous
or even non-homologous nucleic acid sequences; in the context of the present
invention,
oligonucleotides corresponding to families of colon-modified nucleic acids are
shuffled.
The present invention provides significant advantages over previously used
methods for optimization of genes. For example, DNA shuffling of colon
modified nucleic
acids can result in optimization of a desirable property even in the absence
of a detailed
14
CA 02331335 2000-12-21
WO 00/18906 PCT/US99122588
understanding of the mechanism by which the particular property is mediated.
In addition,
entirely new properties can be obtained upon shuffling of codon modified DNAs,
i.e.,
shuffled DNAs can encode polypeptides or RNAs with properties entirely absent
in the
parental DNAs which are shuffled. Thus, by modifying the codon usage and/or
encoded
amino acids of the relevant gene or other nucleic acid, molecular diversity is
accessed and
sequences can be shuffled to obtain desired, including entirely new,
properties.
In general, sequence recombination can be achieved in many different formats
and permutations of formats, as described in further detail below.
The targets for modification vary in different applications, as does the
property sought to be acquired or improved. Examples of candidate targets for
acquisition of
a property or improvement in a property include genes that encode proteins
which have
enzymatic or therapeutic or other commercially useful activities. A more
extensive listing is
found supra; however, even this list is not intended to be limiting, as
essentially any nucleic
acid can be codon modified and shuffled, using one or more of the processes
herein.
I S Shuffling methods use at least two variant forms of a starting target (the
variant forms can be nucleic acids, or representations thereof, e.g., as
character strings in a
computer program). The variant forms of candidate codon-altered substrates can
show
substantial sequence or secondary structural similarity with each other, but
they should also
differ in at least one and preferably at least two positions. The initial
diversity between forms
can be the result of natural variation, e.g., the different variant forms
(homologs) are obtained
from different individuals or strains of an organism, or constitute related
sequences from the
same organism (e.g., aileIic variations), or constitute homologs from
different organisms
(interspecific variants), or constitute artificial homologs, e.g., codon-
altered nucleic acids
encoding the same or a similar protein. Any or all of these sequences can
represent or
include codon altered nucleic acids.
Initial diversity can also be induced, e.g., the variant forms can be
generated
by error-prone transcription, such as an error-prone PCR or use of a
polymerase which lacks
proof reading activity (see, Liao (1990) Gene 88:107-111), of the first
variant form, or, by
replication of the first form in a mutator strain (mutator host cells are
discussed in further
detail below, and are generally well known). The initial diversity between
substrates is
greatly augmented in subsequent steps of recombination for library generation.
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/ZZ588
A mutator strain can include any mutants in any organism impaired in the
functions of mismatch repair. These include mutant gene products of mutS,
mutT, mutes,
mutt, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. The impairment is achieved
by genetic
mutation, allelic replacement, selective inhibition by an added reagent such
as a small
compound or an expressed antisense RNA, or other techniques. Impairment can be
of the
genes noted, or of homologous genes in any organism. The properties or
characteristics that
can be acquired or improved vary widely, and, of course depend on the choice
of substrate.
At least two variant forms of a nucleic acid, e.g., which can confer a desired
activity or which can be recombined to produce a desired activity, are
recombined to produce
a library of recombinant nucleic acids. The library is then screened to
identify at least one
recombinant nucleic acid that is optimized for the particular property or
properties of interest.
Often, improvements are achieved after one round of recombination and
selection. However, recursive sequence recombination can be employed to
achieve still
further improvements in a desired property, or to bring about new (or
"distinct") properties.
Recursive sequence recombination entails successive cycles of recombination to
generate
molecular diversity. That is, one creates a family of nucleic acid molecules
showing some
sequence identity to each other but differing due to the presence of
mutations. In any given
cycle, recombination can occur in vivo or in vitro, intracellularly or
extracellularly.
Furthermore, diversity resulting from recombination can be augmented in any
cycle by
applying known methods of mutagenesis (e.g., error-prone PCR or cassette
mutagenesis) to
either the substrates or products for recombination. In general, however, a
single cycle of
DNA shuffling of codon-altered nucleic acids provides for generation of
surprisingly
effective nucleic acids. Accordingly, while recursive approaches to shuffling
can be used,
single cycle recombination is also preferred. Typically, 2, 3, 4, 5, or even
10 or more cycles
of recombination can be performed, each cycle optionally comprising one or
more selection
steps.
A recombination cycle is usually followed by at least one cycle of screening
or
selection for molecules having a desired property or characteristic. If a
recombination cycle
is performed in vitro, the products of recombination, i.e., recombinant
segments, are
sometimes introduced into cells before the screening step. Recombinant
segments can also
be linked to an appropriate vector or other regulatory sequences before
screening.
16
CA 02331335 2000-12-21
WO 00/1$906 PCT/US99/225$8
Alternatively, products of recombination generated in vitro are sometimes
packaged in
viruses (e.g., bacteriophage) before screening. If recombination is performed
in vivo,
recombination products can sometimes be screened in the cells in which
recombination
occurred. In other applications, recombinant segments are extracted from the
cells, and
optionally packaged as viruses, before screening.
The nature of screening or selection depends on what property or
characteristic is to be acquired or the property or characteristic for which
improvement is
sought, and many examples are discussed below. It is not usually necessary to
understand the
molecular basis by which particular products of recombination (recombinant
segments) have
acquired new or improved properties or characteristics relative to the
starting substrates. For
example, a gene can have many component sequences, each having a different
intended role
(e.g., coding sequences, regulatory sequences, targeting sequences, stability-
conferring
sequences, subunit sequences and sequences affecting integration). Each of
these component
sequences can be varied and recombined simultaneously. Screening/selection can
then be
1 S performed, for example, for recombinant segments that have increased
ability to confer
activity upon a cell without the need to attribute such improvement to any of
the individual
component sequences of the vector.
Depending on the particular screening protocol used for a desired property,
initial rounds) of screening can sometimes be performed using bacterial cells
due to high
transfection efficiencies and ease of culture. However, bacterial expression
is often not
practical or desired, and yeast, fungal or other eukaryotic systems are also
used for library
expression and screening. Similarly, other types of screening which are not
amenable to
screening in bacterial or simple eukaryotic library cells, are performed in
cells selected for
use in an environment close to that of their intended use. Final rounds of
screening can be
performed in the precise cell type of intended use.
If further improvement in a property is desired, at least one and usually a
collection of recombinant segments surviving a first round of
screening/selection are subject
to a further round of recombination. These recombinant segments can be
recombined with
each other or with exogenous segments representing the original substrates or
further variants
thereof. Again, recombination can proceed in vitro or in vivo. If the previous
screening step
identifies desired recombinant segments as components of cells, the components
can be
l7
CA 02331335 2000-12-21
WO 00/18906 PCTNS99l22588
subjected to further recombination in vivo, or can be subjected to further
recombination in
vitro, or can be isolated before performing a round of in vitro recombination.
Conversely, if
the previous screening step identifies desired recombinant segments in naked
form or as
components of viruses, these segments can be introduced into cells to perform
a round of in
vivo recombination. The second round of recombination, irrespective how
performed,
generates further recombinant segments which encompass additional diversity
that is present
in recombinant segments resulting from a previous round (or from multiple
previous rounds,
e.g., where the process is iteratively repeated).
The second round of recombination can be followed by a further round of
screening/selection according to the principles discussed above for the first
round. The
stringency of screening/selection can be increased between rounds. Also, the
nature of the
screen and the property being screened for can vary between rounds if
improvement in more
than one property is desired or if acquiring more than one new property is
desired.
Additional rounds of recombination and screening can then be performed until
the
recombinant segments have sufficiently evolved to acquire the desired new or
improved
property or function.
The practice of this inventian involves the construction of recombinant
nucleic
acids and the expression of genes in transfected host cells. Molecular cloning
techniques to
achieve these ends are known in the art. A wide variety of cloning and in
vitro amplification
methods suitable for the construction of recombinant nucleic acids such as
expression vectors
are well-known to persons of skill. General texts which describe molecular
biological
techniques useful herein, including mutagenesis, include Berger and Kimmel,
Guide to
Molecular Cloning Technigues, Methods in Enzymology volume 152 Academic Press,
Inc.,
San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory
Manual (2nd
Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York,
1989
("Sambrook") and Current Protocols in Molecular Bioloav, F.M. Ausubel et al.,
eds., Current
Protocols, a joint venture between Greene Publishing Associates, Inc. and John
Wiley &
Sons, Inc., (supplemented through 1998) ("Ausubel")). Methods of transducing
cells,
including plant and animal cells, with nucleic acids are generally available,
as are methods of
expressing proteins encoded by such nucleic acids. In addition to Bergen
Ausubel and
Sambrook, useful general references for culture of animal cells include
Freshney (Culture of
18
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
Animal Cells, a Manual of Basic Techniaue, third edition Wiley- Liss, New York
( 1994)) and
the references cited therein, Humason (Animal Tissue Techniques, fourth
edition W.H.
Freeman and Company (1979)) and Ricciardelli, et al., In Vitro CeII Dev. Biol.
25:1016-1024
( 1989). References for plant cell cloning, culture and regeneration include
Payne et al.
(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc.
New York,
NY (Payne); and Gamborg and Phillips (eds) ( 1995) Plant Cell. Tissue and
Organ Culture:
Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg
New York)
(Gamborg). A variety of Cell culture media are described in Atlas and Parks
(eds) The
Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL (Atlas).
Additional
information for plant cell culture is found in available commercial Literature
such as the Life
Science Research Cell Culture Catalogue ( 1998) from Sigma- Aldrich, Inc {St
Louis, MO)
(Sigma-LSRCCC) and, e.g., the Plant Culture Catalogue and supplement (1997)
also from
Sigma-Aldrich, Inc (St Louis, MO) (Sigma-PCCS).
Examples of techniques sufficient to direct persons of skill through in vitro
1 S amplification methods, including the polymerase chain reaction (PCR), the
ligase chain
reaction (LCR), Q(J-replicase amplification and other RNA polymerase mediated
techniques
(e.g., NASBA) are found in Berger, Sambrook, and Ausubel, id., as well as in
Mullis et al.,
(1987) U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and
Applications
(Innis et al. eds) Academic Press Inc. San Diego, CA ( 1990) (Innis); Arnheim
& Levinson
(October l, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94;
Kwoh et al.
{ 1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. ( 1990) Proc.
Natl. Acad. Sci.
USA 87, 1874; Lomell et al. ( 1989) J. CIin. Chem 35, 1826; Landegren et al.,
( 1988) Science
241, 1077-1080; Van Brunt (1990) BiotechnoloQV 8, 291-294; Wu and Wallace,
(1989) Gene
4, 560; Barringer et al. ( 1990) Gene 89, 117, and Sooknanan and Malek ( 1995
)
Biotechnolo~y 13: 563-564. Improved methods of cloning in vitro amplified
nucleic acids
are described in Wailace et al., U.S. Pat. No. 5,426,039. Improved methods of
amplifying
large nucleic acids by PCR are summarized in Cheng et al. ( 1994) Nature 369:
684-685 and
the references therein, in which PCR amplicons of up to 40kb are generated.
One of skill will
appreciate that essentially any RNA can be converted into a double stranded
DNA suitable
for restriction digestion, PCR expansion and sequencing using reverse
transcriptase and a
poiymerase. See, Ausbel, Sambrook and Berger, all supra.
19
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
Oligonucleotides e.g., for use in in vitro amplification/ gene reconstruction
methods, for use as gene probes, or as shuffling targets (e.g., synthetic
genes or gene
segments) are typically synthesized chemically according to the solid phase
phosphoramidite
triester method, e.g., as described by Beaucage and Caruthers (1981),
Tetrahedron Letts.,
22(20):1859-1862, e.g., using an automated synthesizer, e.g., as described in
Needham-
VanDevanter et al. ( 1984) Nucleic Acids Res., 12:6159-6168 or as is now
practiced routinely
in the art. Oligonucleotides can also be custom made and ordered from a
variety of
commercial sources known to persons of skill. Purification of oligonucleotides
(e.g., using
gel-purification methods) to improve the quality of synthesized
oligonucleotides can be
I O particularly desirable in the processes herein to improve the quality of
nucleic acid synthesis
protocols.
As noted, essentially any nucleic acid can be custom ordered from any of a
variety of commercial sources, such as The Midland Certified Reagent Company
(mcrc@oligos.com), The Great American Gene Company (http://www.genco.com),
1 S ExpressGen Inc. (www.expressgen.com), Operon Technoloigies Inc. (Alameda,
CA) and
many others. Similarly, peptides and antibodies can be custom ordered from any
of a variety
of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, inc.
(http://www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio~Synthesis, Inc., and
many
others.
20 CODON AND AMINO ACID ALTERED LIBRARIES
In the methods of the invention, libraries of codon altered nucleic acids can
be
made and recombined. The codon altered nucleic acids can also include
differences in
encoded amino acid sequences, which can be either conservative or non-
conservative in
nature. The codon altered nucleic acids can be derived from a single parental
amino acid
25 sequence, or can be derived from a family of original sequences, e.g.,
natural or synthetic
homologous variants of a given sequence. Libraries can exist, e.g., in pools
or aliquots of
cells, viral plaques, enzyrnatically synthesized pools or aliquots of nucleic
acids, or
chemically synthesisized pools of nucleic acids. Methods of making libraries
of nucleic acids
are available and taught, e.g., in Berger, Sambrook and Ausubel, supra. In one
embodiment,
30 a library as used in the invention comprises at least 2 nucleic acid
sequences. In additional
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
embodiments, the libraries of this invention comprise at least 2, S, 10, 100,
1000, or more
nucleic acid sequences.
As applied to the invention, libraries are typically constructed with a high
percentage of codons altered relative to an initial (e.g., wild type) nucleic
acid. Codon usage
S divergence for each of the codon altered nucleic acids can be SO%, 7S%, or
even 90% or
more as compared to the first nucleic acid. This eliminates hybridization to
the parental
nucleic acid (and thereby inhibits recombination with the parental nucleic
acid, a desirable
feature in certain embodiments discussed below).
In several embodiments of this invention, codons are modified in members of
a gene family so as to increase the degree of identity between the members. In
one such
embodiment, the genes are homologous genes from different species. In such
cases, the
degree of nucleic acid identity may be lower than the degree of amino acid
identity, at least in
part, because of differences in codon usage between the species. In additional
embodiments,
the homologous genes represent different members of a gene family within a
single species.
1 S Such genes may encode functionally distinct members of a gene family that
nevertheless
share significant structural or functional similarity. In preferred
embodiments, homologous
genes are reverse translated into nucleic acid sequences, and the nucleic acid
sequences are
modified so as to increase the level of identity between them. Nucleic acids
with the
modified sequences can then be synthesized in vitro. In particularly preferred
embodiments,
the modified nucleic acid sequences are at least as identical to each other as
the original
amino acid sequences.
Additional sequence diversity is provided by generating nucleic acids with
non-overlapping non-conservative substitutions in each of the codon altered
nucleic acids as
compared to the first nucleic acid. This provides for reversion to wild-type
upon
2S recombination, while optionally allowing for the incorporation of non-
conservative changes
to the sequence in the event that they produce a detectable improvement during
screening.
Modification of the codons of one or more of the codon altered nucleic acids
to provide one or more different hydrophobic core residue for an encoded
polypeptide as
compared to the first polypeptide is also provided. This modification of core
amino acids
provides minor differences in encoded proteins, while changing the mutational
spectrum of
the resulting nucleic acid, thereby increasing sequence diversity.
21
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
In addition, due to the constraints of the translational machinery for a given
cell, codon usage may need to be altered when expressed sequences are shuttled
between
different organisms (e.g., animal cells, plant cells, bacterial cells, etc.)
for optimal expression.
This produces a nucleic acid which encodes the same protein, but which, after
typical forms
S of point mutation, will access a different mutational diversity than the
original form of the
protein.
In one embodiment, phage libraries are made and recombined in mutator
strains such as cells with mutant or impaired gene products of mutS, mutT,
mutes, mutt,
ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. The impairment is achieved by
genetic
mutation, allelic replacement, selective inhibition by an added reagent such
as a small
compound or an expressed antisense RNA, or other techniques. High multiplicity
of
infection (MOI) libraries are used to infect the cells to increase
recombination frequency.
Additional strategies for making phage libraries and or for recombining DNA
from donor and
recipient cells are set forth in U.S. Pat. No. S,S21,077. Additional
recombination strategies
1 S for recombining plasmids in yeast are set forth in WO 97 07205.
The library to be made can be an in vitro set of molecules, or present in
cells,
phage or the like. Virtual libraries of nucleic acids generated in silico are
also a feature of the
invention (see also, Selifonov and Stemmer, supra). Generally, the library is
screened to
identify at least one recombinant nucleic acid that exhibits distinct or
improved activity
compared to the parental nucleic acid or nucleic acids which are recombined.
Additional
details on making appropriate libraries are found below, e.g., in the section
entitled "Formats
for Sequence Recombination."
TARGETS FOR CODON MODIFICATION AND SHUFFLING
Essentially any nucleic acid can be codon altered and shuffled. No attempt is
2S made herein to identify the hundreds of thousands of known nucleic acids.
Common
sequence repositories for known proteins include GenBank EMBL, DDBJ and the
NCBI.
Other repositories can easily be identified by searching the Internet.
One class of preferred targets for activation includes nucleic acids encoding
therapeutic proteins such as erythropoietin (EPO), insulin, peptide hormones
such as human
growth hormone; growth factors and cytokines such as epithelial Neutrophil
Activating
Peptide-78, GROa/MGSA, GROG, GROy, MIP-la, MIP-1(3, MCP-l, epidermal growth
22
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
factor, fibroblast growth factor, hepatocyte growth factor, insulin-like
growth factor, the
interferons, the interleukins, keratinocyte growth factor, leukemia inhibitory
factor,
oncostatin M, PD-ECSF, PDGF, pleiotropin, SCF, c-kit ligand, VEGEF, G-CSF etc.
Many
of these proteins are commercially available (See, e.g., the Sigma BioSciences
1997
catalogue and price list), and the corresponding genes are well-known.
Another class of preferred targets are transcriptional and expression
activators.
Example transcriptional and expression activators include genes and proteins
that modulate
cell growth, differentiation, regulation, or the like. Expression and
transcriptional activators
are found in prokaryotes, viruses, and eukaryotes, including fungi, plants,
and animals,
including mammals, providing a wide range of therapeutic targets. It will be
appreciated that
expression and transcriptional activators regulate transcription by many
mechanisms, e.g., by
binding to receptors, stimulating a signal transduction cascade, regulating
expression of
transcription factors, binding to promoters and enhancers, binding to proteins
that bind to
promoters and enhancers, unwinding DNA, splicing pre-mRNA, polyadenylating
RNA, and
degrading RNA. Expression activators include cytokines, inflammatory
molecules, growth
factors, their receptors, and oncogene products, e.g., interleukins (e.g., IL-
1, IL-2, IL-8, etc.),
interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-a, TGF-Vii, EGF, KGF,
SCF/c-Kit,
CD40L/CD40, VLA-4/VCAM-I, ICAM-1/LFA-I, and hyalurin/CD44; signal transduction
molecules and corresponding oncogene products, e.g., Mos, Ras, Raf, and Met;
and
transcriptional activators and suppressors, e.g., p53, Tat, Fos, Myc, Jun,
Myb, Rel, and
steroid hormone receptors such as those for estrogen, progesterone,
testosterone, aldosterone,
the LDL receptor ligand and corticosterone.
Similarly, proteins from infectious organisms for possible vaccine
applications, described in more detail below, including infectious fungi,
e.g., Aspergillus,
Candida species; bacteria, particularly E. toll, which serves a model for
pathogenic bacteria,
as well as medically important bacteria such as Staphylococci (e.g., aureus),
Streptococci
(e.g., pneumoniae), Clostridia (e.g., perfringens), Neisseria (e.g.,
gonorrhoea),
Enterobacteriaceae (e.g., toll), Helicobacter (e.g., pylori), Vibrio (e.g.,
cholerae),
Capylobacter (e.g., jejuni), Pseudomonas (e.g., aeruginosa), Haemophilus
(e.g., influenzae),
Bordetella (e.g., pertussis), Mycoplasma (e.g., pneumoniae), Ureaplasma (e.g.,
urealyticum),
Legionella (e.g., pneumophila), Spirochetes (e.g., Treponema, Leptospira, and
Borrelia),
23
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
Mycobacteria (e.g., tuberculosis, smegmatis), Actinomyces (e.g., israelii),
Nocardia (e.g.,
asteroides), Chlamydia {e.g., trachomatis), Rickettsia, Coxiella, Ehrilichia,
Rochalimaea,
Brucella, Yersinia, Fracisella, and Pasteurella; protozoa such as sporozoa
(e.g., Plasmodia),
rhizopods (e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania,
Trichomonas,
Giardia, etc.); viruses such as ( + ) RNA viruses (examples include Poxviruses
e.g., vaccinia;
Picornaviruses, e.g. polio; Togaviruses, e.g., rubella; Flaviviruses, e.g.,
HCV; and
Coronaviruses), ( - ) RNA viruses (examples include Rhabdoviruses, e.g., VSV;
Paramyxovimses, e.g., RSV; Orthomyxovimses, e.g., influenza; Bunyaviruses; and
Arenaviruses), dsDNA viruses (Reoviruses, for example), RNA to DNA viruses,
i.e.,
Retroviruses, e.g., especially HIV and HTLV, and certain DNA to RNA viruses
such as
Hepatitis B virus.
Other proteins relevant to non-medical uses, such as inhibitors of
transcription
or toxins of crop pests e.g., insects, fungi, weed plants, and the like, are
also preferred targets
for shuffling. Industrially important enzymes such as monooxygenases,
proteases, nucleases,
and lipases are also preferred targets. As an example, subtilisin can be
evolved by shuffling
codon altered forms of the gene for subtilisin (von der Osten et al., J.
Biotechnol. 28:55-68
(1993) provide a subtilisin coding nucleic acid). Proteins which aid in
folding such as the
chaperonins are also preferred.
Preferred known genes suitable for codon alteration and shuffling also include
the following: Alpha-1 antitrypsin, Angiostatin, Antihemolytic factor,
Apolipoprotein,
Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial
peptides, C-X-C
chemokines (e.g., T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2,
NAP-4,
SDF-1, PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractant
protein-1,
Monocyte chemoattractant protein-2, Monocyte chemoattractant protein-3,
Monocyte
inflammatory protein-1 alpha, Monocyte inflammatory protein-I beta, RANTES,
I309,
883915, 891733, HCC1, T58847, D31065, T64262), CD40 ligand, Collagen, Colony
stimulating factor (CSF), Complement factor Sa, Complement inhibitor,
Complement
receptor 1, Factor IX, Factor VII, Factor VIII, Factor X, Fibrinogen,
Fibronectin,
Glucocerebrosidase, Gonadotropin, Hedgehog proteins (e.g., Sonic, Indian,
Desert),
Hemoglobin (for blood substitute; for radiosensitization), Hirudin, Human
serum albumin,
Lactoferrin, Luciferase, Neurturin, Neutrophil inhibitory factor (NIF),
Osteogenic protein,
24
CA 02331335 2000-12-21
WO OO/I8906 PCTNS99/22588
Parathyroid hormone, Protein A, Protein G, Relaxin, Renin, Salmon calcitonin,
Salmon
growth hormone, Soluble complement receptor I, Soluble I-CAM 1, Soluble
interleukin
receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, I5), Soluble TNF
receptor,
Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigens, i.e.,
Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), Toxic
shock
syndrome toxin (TSST-1), Exfoliating toxins A and B, Pyrogenic exotoxins A, B,
and C, and
M. arthritides mitogen, Superoxide dismutase, Thymosin alpha 1, Tissue
plasminogen
activator, Tumor necrosis factor beta (TNF beta), Tumor necrosis factor
receptor (TNFR),
Tumor necrosis factor-alpha (TNF alpha) and Urokinase. Many other known coding
nucleic
acids, such as those in GenebankTM, can be codon-altered and shuffled.
GENES WITH CODON USAGE REDESIGNED AND CHEMICALLY SYNTHESIZED
AS STARTING MATERIALS FOR GENE FAMILY SHUFFLING--EXPANDING THE
DIVERSITY OF DNA SHUFFLING.
Because the genetic coding preference among organisms ranges from quite
similar to very different, homologous genes from different organisms can have
significantly
lower homology at the nucleic acid level than at the amino acid level. For
example, genetic
information for some bacterial species is high in GC content (up to 70%),
while others have
AT rich (>60%) codon usage. Thus, genes from different organisms rnay have,
for example,
40-60% amino acid identity but only 25-35% nucleic acid identity. It is often
desirable to
increase such levels of nucleic acid identity so as to enhance the ability of
the homologous
sequences to recombine, thereby increasing the efficiency of family shuffling
using the
methods of this invention. In other aspects, it is actually preferable to
decrease the rate of
recombination in a system, e.g., when using vectors it is sometimes desirable
to decrease the
rate of recombination between the vector and the host DNA, thereby increasing
the safety of
the vector. The following examples address specific issues with regard to
shuffling codon
altered nucleic acids.
Altering codon usage to increase homology
In one aspect, protein sequences of gene family members are reverse
translated back into DNA sequences, for example by using one of the preferable
codon usage
charts in any conventional DNA manipulation program (e.g. the Wisconsin
PackageTM,
SeqWeb, OMIGA, SeqApp, SeqPup, MacVector, DNA stryder, GeneWorks, etc.). The
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
choice of codon usage is often determined by the host in which the genes will
be expressed.
After maximizing the percentage of DNA sequence identity, the genes are
chemically
synthesized, e.g., using a high throughput oligonucleotide synthesizer in,
e.g. a 96-well
format, optionally in conjunction with polymerase and/ or ligase gene
synthesis methods.
In general, the DNA sequence similarity after such treatment will be at least
as
high as the amino acid similarity, but can be at least about 10% to 15% higher
than the amino
acid identity (in contrast to the situation for naturally occurring genes,
which are ordinarily
less well conserved than encoded polypeptides), based on the random frequency
of sequence
identity for any given codon. In most cases, the minimal requirement for amino
acid identity
can be as low as about 35% while still retaining adequate nucleic acid
homology for standard
recombination methods (as discussed, supra, oligonucleotide-mediated
recombination
methods do not require high levels of similarity to achieve recombination). In
some cases,
however, the minimal amino acid identity can be even lower, e.g. if the
conserved regions are
clustered within the genes.
i 5 Example: Shuffling codon-modified EPO
The protein erythropoietin alpha, also known as EPO, Epogen, and Procrit is a
hematopoietic hormone, providing a variety of benefits to patients suffering
from anemia (a
common symptom of, e.g., AIDS). EPO is produced as a pharmaceutical, with
sales of
nearly 1 billion dollars world-wide. Accordingly, proteins with EPO-like
activity (and
preferably superior activity) are of substantial commercial interest.
Figure 1 shows the sequence of a part of the monkey EPO gene, which is
similar to the human EPO gene. Figure 2 shows an example of a codon altered
EPO nucleic
acid (or "wobble" EPO gene). In general, transversions rather than transition
mutations are
made where possible. The purpose of this strategy is to maximally disrupt
hybridization of
the resulting gene with naturally occurring EPOS. Figure 3 shows an alignment
of naturally
occurring EPOS.
This strategy is further fine-tuned by applying standard rules of base pairing
(e.g., elimination of G-C pairing and GC stacking) to maximize sequence
disruption; in
addition, conservative or non-conservative amino acid modifications can also
be made (in
some cases, where multiple codon-altered nucleic acids are shuffled, it is
desirable to make
codon altered nucleic acids with non-overlapping non-conservative
substitutions to permit
26
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
reversion to the wild-type amino acid during shuffling). The size of the
sequence space for
nucleic acids encoding EPO is large, at about 2.8 x 1088 different sequences
(there are about
10$° particles in the universe; thus, it is physically impossible to
make all of the possible
sequences encoding EPO). As indicated schematically in Figure 4, if one only
considers the
maximally divergent wobble genes (those that use the alternative types of
colons for leucine,
arginine, and serine), there is still a sequence space of 1038 sequences
encoding EPO. The
overall strategy is to synthesize a library of wobble genes, screen for
expression and activity
and DNA shuffle desirable genes as desired (e.g., by recursive processes).
It is of interest to further evolve colon-altered nucleic acids. Shuffling
with
other homologous genes from nature, designed genes (incorporating libraries of
designed
sequence variation), and genes containing mutations of interest are strategies
for evolving
any gene of interest. However, the colon altered nucleic acid may not be
easily shuffled with
these genes because of the sequence differences; or they may be undesirable
for other reasons
(e.g., the naturally occurring sequences may be proprietary, or include
proprietary elements).
These difficulties can be avoided by synthesizing colon-altered homologous
nucleic acids
which encode desired amino acid variations (e.g., those found in homologous
genes), but
which have a colon-set close to the nucleic acids) to be recombined (thereby
permitting,
e.g., hybridization during recombination).
For example, after identifying homologues of interest (e.g., those shown in
Figure 3 for EPO), colon-altered nucleic acids encoding the same proteins are
synthesized
with a similar colon selection. Standard family shuffling is then practiced
with the colon
altered nucleic acids. This is shown schematically for EPO in Figure S.
EPO wobble variants are screened for expression and then receptor binding
assays are conducted in an ELISA format, using human EPOr-Fc fusions.
Following
selection of binding variants, activity is measured as thymidine incorporation
in UT7-EPO (A
human bone marrow cell line) cell proliferation assays. Cells are treated for
2-3 days with
various concentration of EPO variants after which time they are incubated in
the presence of
3-H thymidine for 4 hours and incorporation of thymidine is measured. See
also, Erickson-
miller et al. (1997) Blood 90:2421 (for the receptor binding assay), and Wen
et al. (1994) J.
Biol. Chem. 269:22839-22846 (for the thymidine incorporation assay).
27
CA 02331335 2000-12-21
WO 00/18906 PCTNS99/22588
Assays for selecting EPO can also be based, e.g., on the ability of EPO
proteins to stimulate the growth of blood cell, e.g., in vitro or in vivo.
Example: Codon Shuffling G-CSF
Family shuffling can be used to breed diversity from genes into the libraries
to
be screened. Additionally, design heuristics such as randomization of
hydrophobic core
residues can be used to take advantage of the redundancy between primary
structure and
tertiary structure of proteins (i.e. many different primary structures encode
proteins with very
similar three dimensional structures).
Design heuristics are employed to create a sequence space of mutants that are
predicted to be highly biased (relative to random mutagenesis) to encode
proteins which
preserve the original activity. Methods such as high throughput (HTP)
screening and phage
panning are used to identify members of the designed libraries that have the
desired activity.
DNA shuffling is used to breed this population of active clones in order to
fine tune the
mutants, thus allowing one to evolve variants with equivalent or superior
function relative to
the naturally occurring proteins.
Figures 6 and 7 show several mammalian homologues of G-CSF. Figure 8
shows the hydrophobic core residues of human G-CSF (blacked out). Figure 9
shows a
strategy for evolving variants of human G-CSF that are highly divergent in
sequence. First,
three genes are synthesized (Genes 1, 2 and 3, Figure 8) which contain all of
the mammalian
homologue diversity of G-CSF. These genes are shuffled, phage panned against
the G-CSF
receptor, and HTP screened for biological function (receptor activation).
Active clones are
iteratively shuffled and screened if necessary to give evolved variants that
rival or surpass the
human gene in activity (on human cells).
Next, one evolves a variant that has a highly mutated hydrophobic core. This
is schematically illustrated in Figure 8, and the specific strategy for
performing the biological
screening is schematically illustrated in Figure 9. it is expected that the
best mutants
obtained after screening hydrophobic core randomized libraries may be less
active than wild
type human G-CSF because it is difficult to initially optimize activity in
such a procedure.
Family shuffling is used to obtain optimized variants. This is done by
synthesizing genes
which contain mammalian homologue diversity at all but the hydrophobic core
positions; but
28
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
they are synthesized in the context of an evolved, non-wild type hydrophobic
core. Family
shuffling is used to optimize around the new hydrophobic core.
This strategy works because there are functionally similar hydrophobic cores
for wild type proteins that consist of largely different amino acids than the
wild type protein.
This understanding is supported by recent experiments in model systems. For
example, S3%
of randomized sequences for three residues in the hydrophobic core of lambda
repressor are
folded and biologically active (Lim and Sauer ( 1991 ) J. Mol. Biol. 219:359-
376). Protein
design by patterning of polar and non-polar amino acids, where 24 residues in
the
hydrophobic core of a 4-helix bundle protein were randomized by Kamtekar et
al. (1993)
Science 262:1321. Folded, alpha helical proteins were recovered from about 1 %
of the
clones. Desjarlais and Handel ( 1995) Current Opinion in BiotechnoloQV 6:460-
466 showed a
mutant of Rop, another 4-helix bundle protein, where four hydrophobic core
residues have
been randomized and active mutants have been obtained. Axe et al. (1996) PNAS
9S:SS90-
SS94 showed that randomizing 13 hydrophobic core residues in the enzyme
barnase resulted
1 S in 23% of the clones in the library retaining biological activity.
Gassener et al. ( 1996) PNAS
93:12155-12158 describe a mutant of T4 lysozyme where 10 residues in the
hydrophobic
core are replaced with Met. This is taken as evidence that the hydrophobic
core of this
protein is very tolerant to substitution. Taken together, this experimental
evidence on model
systems shows that the hydrophobic cores of many proteins can be replaced with
other
hydrophobic residues that pack in a similar fashion to give an active protein.
This
degeneracy is exploited to evolve novel forms of natural and codon-altered
genes.
A related approach is to search the protein databases for a protein that has a
similar activity to a protein that on wishes to evolve. Denesyuk et ai. {
1996) J. Theor. Biol
shows the results of such a search for G-CSF. LIF is a very similarly folded
protein. One
2S can use LIF as a 'scaffold' on which to place residues of G-CSF that are
required for activity.
Given LIF with a G-CSF "toupee," one would family shuffle the LIF scaffold so
as to obtain
a variant in which the toupee is displayed in a fully biologically active
form.
Another approach is to use computational methods to create families of
variants that are predicted to be functional. Dahiyat and Mayo Science
recently described
computer methods that are used to design proteins. Proteins are simulated on
the computer,
often with the aid of genetic algorithms, and a subset that are deemed 'fit'
are actually
29
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
synthesized and 'analyzed'. These computational methods are becoming
increasingly
powerful. They would be useful to, for example, predict a family of mutations
on the surface
of a protein that would not destroy function. DNA shuffling can be used to
optimized active
clones obtained by design. Taking the example of G-CSF, one could use
computational
methods in combination with all structure function data (for example alanine
scan data for G-
CSF reported recently by Reidhaar-Olsen in Biochemistry) to design a family of
putatively
functional variants. One could, for example, design the family to have minimal
DNA identity
to the wild type gene given the design constraints. This library is
synthesized, put through
biological screens and/or selections (i.e. panning against the G-CSF
receptor), and active
variants are obtained. DNA shuffling is then used to evolve these active
variants to have the
desired level of function.
G-CSF proteins are displayed on phage and screened for binding to human G-
CSF receptor in an ELISA format. Variants that bind receptor are selected in a
high
throughput screen for receptor activation. This cell based assay measures
receptor activation
via a reporter gene (such as luciferase) activated by a G-CSF responsive
construct containing
STAT binding elements. Cells (such as HepG2) are transformed with a G-CSF
responsive
reporter plasmid and treated with the codon shuffled G-CSF variant for 2.5
hours. Cells are
then lysed and luciferase activity measured. See also, Tian et al (1998)
Science 281:257-259.
Example: Codon Shuffling Alkaline Phosphatase
Alkaline phosphatase is a widely used reporter enzyme for ELISA assays,
protein fusion assays, and in a secreted form as a reporter gene for mammalian
cells. A more
active form of the enzyme is desirable.
A codon altered form of alkaline phosphatase was generated by PCR assembly
using the oligos set forth in Figure 10. A map of the oligos is set forth in
Figure 11. The
procedure used was essentially identical to that taught in Stemmer et al. (
1994) Gene 164:49-
57. In brief, the oligos were mixed 1:1 at a variety of dilutions and PCR
assembled by
performing e.g., 25-60 cycles of PCR at e.g., 94 °C (60 sec.), 94
°C (30 sec.), 50 °C (30 sec.),
72 °C (30 sec). Assembly of the BIAP gene was conducted in a circular
format and gene
fragements were purified. 100,000 colonies were screened on LB/am plates 01/10
are wt
plasmid). About 1/10 showed a bluer color than background. Plasmid DNA showed
a
correct insertion.
CA 02331335 2000-12-21
WO 00/18906 PCTNS99/2Z588
In general, petri-dish screening using the typical colorimetric assay for
phosphatase activity can be used for screening. This has the advantage or
being simple, high
throughput, and semi quantitative. Microtiter plate screening, also preferred,
is colorimetric,
and quantitative, although additional instrumentation can be required for
implementation.
Example: Codon Shuffling to Reduce Competent Virus Production from
Vectors and to Generate Attenuated Viruses as Immunogenic Compositions
and Vaccines
Cells can be stably transduced with a number of viral vectors including those
derived from retroviruses, pox viruses, adenoviruses {Ads), herpes viruses and
parvoviruses.
Common viral vectors include those derived from murine leukemia viruses
(MuLV), gibbon
ape leukemia viruses (GaLV), human immuno deficiency viruses (HIV),
adenoviruses, adeno
associated viruses (AAVs), Epstein Barr viruses, canarypox viruses, cowpox
viruses, and
vaccinia viruses. Viral vectors based upon retroviruses, adeno-associated
viruses, herpes
viruses and adenoviruses are all used as gene therapy vectors for the
introduction of
therapeutic nucleic acids into the cells of an organism by ex vivo and in vivo
methods.
When using viral vectors, packaging cells are commonly used to prepare
virions used to transduce target cells. In these vectors, traps-active genes
are rendered
inactive and "rescued" by traps-complementation to provide a packaged vector.
This form of
traps complementation is provided by co-infection of a packaging cell with a
virus or vector
which supplies functions missing from a particular gene therapy vector in
traps, or by using a
cell line {e.g., 293 cells) which have viral components integrated into the
genome of the
packaging cell. For instance, cells transduced with HIV or murine retroviral
proviral
sequences which lack the nucleic acid packaging site produce retroviral traps
active
components, but do not specifically incorporate the retroviral nucleic acids
into the capsids
produced, and therefore produce little or no live virus.
If these transduced "packaging" cells are subsequently transduced with a
vector nucleic acid which lacks coding sequences for retroviral traps active
functions, but
includes a packaging signal, the vector nucleic acid is packaged into an
infective virion. A
number of packaging cell lines useful for MoMLV-based vectors are known in the
art, such
as PA317 (ATCC CR.L 9078) which expresses MoMLV core and envelope proteins
see,
Miller et al. J. Virol. 65:2220-2224 (1991). Carrol et al. (1994) Journal of
virology
6$(9):6047-6051 describe the construction of packaging cell lines for HIV
viruses.
31
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
Reciprocal complementation of defective HIV molecular clones is described,
e.g., in Lori et
al. (1992) Journal of Virolo~v 66(9) 5553-5560.
Functions of viral replication not supplied by trans-complementation which
are necessary for replication of the vector are present in the vector. In HIV,
this typically
S includes, e.g., the TAR sequence, the sequences necessary for HIV packaging,
the RRE
sequence if the instability elements of the p17 gene of gag is included, and
sequences
encoding the polypurine tract. HIV sequences that contain these functions
include a portion
of the 5' long terminal repeat (LTR) and sequences downstream of the S' LTR
responsible for
efficient packaging, i.e., through the major splice donor site ("MSD"), and
the polypurine
tract upstream of the 3' LTR through the U3R section of the 3' LTR. The
packaging site (psi
site or yr site) is partially located adjacent to the S' LTR, primarily
between the MSD site and
the gag initiator codon (AUG) in the leader sequence. See, Garzino-Demo et al.
( 1995) Hum.
Gene Ther. 6(2): 177-184. For a general description of the structural elements
of the HiV
genome, see, Holmes et al. PCT/EP92/02787.
Another common vector is based upon adenovirus. Typically, vectors which
include the adenovirus ITRs (Gingeras et al. ( 1982) J. Biol. Chem. 257:13475-
13491 ) are
packaged in, e.g., 293 cells, which provide many of the components necessary
for vector
packaging.
Adeno-associated viruses (AAVs) utilize helper viruses such as adenovirus or
herpes virus to achieve productive infection. In the absence of helper virus
functions, AAV
integrates (site-specifically) into a host cell's genome, but the integrated
AAV genome has no
pathogenic effect. The integration step allows the AAV genome to remain
genetically intact
until the host is exposed to the appropriate environmental conditions (e.g., a
lytic helper
virus), whereupon it re-enters the lytic life-cycle. Samulski (1993) Current
Opinion in
Genetic and Development 3:74-80 and the references cited therein provides an
overview of
the AAV life cycle. For a general review of AAVs and of the adenovirus or
herpes helper
functions see, Berns and Bohensky ( 1987) Advanced in Virus Research, Academic
Press.,
32:243-306. The genome of AAV is described in Laughlin et al. ( 1983) Gene,
23:65-73.
Expression of AAV is described in Beaton et al. ( 1989) J. Virol., 63:4450-
4454. In general,
the packaging sites for all parvoviruses, including B 19 and AAV are located
in the viral
ITRs. Recombinant AAV vectors (rAAV vectors) deliver foreign nucleic acids to
a wide
32
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
range of mammalian cells (Hermonat & Muzycka ( 1984) Proc Natl Acad Sci USA
81:6466-
6470; Tratschin et al. ( 1985) Mol Cell Biol 5:3251-3260), integrate into the
host chromosome
(McLaughlin et al. ( 1988) J Virol 62: 1963-1973), and show stable expression
of the
transgene in cell and animal models (Flotte et al. (1993) Proc Natl Acad Sci
USA 90:10613-
10617). rAAV vectors are able to infect non-dividing cells (Podsakoff et al. (
1994) J Virol
68:5656-66; Flotte et al. ( 1994) Am. J. Respir. Cell Mol. Biol. 11:517-521 ).
Further
advantages of rAAV vectors include the lack of an intrinsic strong promoter,
thus avoiding
possible activation of downstream cellular sequences, and the vector s naked
icosohedral
capsid structure, which renders the vectors stable and easy to concentrate by
common
laboratory techniques.
One problem with previously existing vector packaging strategies is that
vectors to be packaged can recombine with nucleic acids providing packaging
functions in
trans, producing a replication-competent virus. This can be a problem both
when vectors are
produced for therapeutic applications (e.g., in gene therapy) and during
production of
encoded components in vitro. The present invention provides a way of reducing
or
eliminating recombination between nucleic acids encoding trans-active
components and
vector nucleic acids encoding packaging sites.
In particular, nucleic acid subsequences of a vector which are adjacent to
modified or deleted elements provided in trans, are codon modified to
eliminate hybridization
to wild-type sequences. Because these sequences do not hybridize, they cannot
recombine
with nucleic acids producing trans-active components. One additional advantage
of this
approach is that the vectors also cannot recombine with live viruses, e.g., in
a human body
which is infected with a virus that packages vector elements. As noted above,
two types of
gene therapy vectors are those based upon retroviruses (which can be packaged
by, e.g., HIV-
1 ) and adenoviruses (which can be packaged by adenovirus).
Alternatively, the nucleic acids encoding trans-active components can be
codon modified so that they do not hybridize to wild-type sequences. This also
prevents
recombination with vectors having wild-type sequences, preventing
recombination and
formation of replication competent viruses.
33
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
After codon modification, vectors or trans active nucleic acids can be
shuffled
as described supra, and screened for the ability to package nucleic acids, or
to be packaged,
as appropriate.
It will be appreciated that codon modification of viral sequences has an
additional use as well. Codon alteration of viral sequences can result in
attenuation of the
virus, e.g., due to modification of regulatory sequences, alterations in mRNA
secondary
structure, inefficient translation due to rare codon use, and the like. Such
"codon attenuated"
viruses have a significant advantage over existing attenuated viruses (which
are typically
generated by serial passage in cells other than the normal host type for the
virus). In
particular, codon attenuated viruses can encode a wild-type set of proteins,
making them
ideal as immunogenic compositions to generate antibodies, or to use as
vaccines. Viral
proteins can also be used in various diagnostic assays. For example, the
standard diagnostic
test for HIV infection in current use tests for the presence of anti-HIV
antibodies in blood by
probing with viral proteins.
Example: Codon Usage Libraries to evolve Functional Variants with reduced
Recombination With Natural Gene Sequences--Adenovirus
Adenovirus is a common vector used, e.g., for gene therapy. The virus is
typically modified to make it replication deficient. This can be achieved
e.g., by deleting the
E 1 and E4 genes. The functions of E 1 and E4 can be supplied by trans
complementation
when E1 and E4 deleted vectors are grown in the ubiquitous human embryonic
kidney cell
line 293, which has uncharacterized adenovirus fragments incorporated into
their genome
that supply the missing functions in trans. The replication defective
adenoviral vectors
recombine at a low, but clinically significant frequency, resulting in
replication competent
adenovirus contamination of vector preparations. Because adenovirus has
detrimental effects
on health, this is a significant problem for application of adenovirus-based
gene therapy
vectors.
In the present invention, a codon usage library encompassing several hundred
bases to several kilobases of sequence flanking the adenovirus E 1 and E4
genes are made.
The library is designed to enforce a high degree of divergence from the
natural adenoviral
consensus sequence, while at the same time incorporating a large degree of
degeneracy in the
codons to allow for a large space of sequence diversity to be searched. The
design principle
34
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
is to obtain mutants that encode the same or similar protein sequence, but
with many
mismatches to the wild-type El and E4 sequences found in the 293 genome. These
mismatches strongly reduce the frequency of unwanted recombination with the
trans
complementary genes. Consequently, engineered adenoviral vectors, or
adenovirus helper
vectors which package adenoviral sequences which include packaging sequences
(adenoviral
or adeno-associated viral ITRs) in trans have reduced levels of recombination.
This provides
for a lower rate of competent adenovirus production, making culture and
production of such
vectors safer.
Evolution Imaaired Viruses Created by Massive Codon Usa~ Alteration As a
General Approach to Vaccines--HIV
HIV-1 and HIV-2 are genetically related, antigenically cross reactive, and
share a common cellular receptor (CD4). See, Rosenburg and Fauci (1993) in
Fundamental
Immunology, Third Edition Paul (ed) Raven Press, Ltd., New York (Rosenburg and
Fauci 1 )
and the references therein for an overview of HIV infection. HIV-1 infection
is epidemic
1 S world wide, causing a variety of immune system-failure related phenomena
commonly
termed acquired immune deficiency syndrome (AIDS). HIV type 2 (HIV-2) has been
isolated from both healthy individuals and patients with AIDS-like illnesses
(Andreasson, et
al. (1993) Aids 7, 989-93; Clavel, et al. (1986) Nature, 324, 691-695; Gao, et
al. (1992)
Nature 358, 495-9; Harnson, et al. ( 1991 ) Journal of Acguired Immune
Deficiency
Syndromes 4, 1155-60; Kanki, et al. ( 1992) American Journal of Epidemiology
136, 895-907;
Kanki, et al. ( 1991 ) Aids Clinical Review 1991, 17-3$; Romieu, et al. (
1990) Journal of
Acquired Immune Deficiency Syndromes 3, 220-30; Naucler, et al. ( 1993)
International
Journal of STD and Aids 4, 217-21; Naucler, et al. ( 1991 ) Aids S, 301-4).
Although HIV-2
AIDS cases have been identified principally from West Africa, sporadic HIV-2
related AIDS
cases have also been reported in the United States (O'Brien, et al. ( 1991 )
Aids 5, 85-8) and
elsewhere. HIV-2 will likely become endemic in other regions over time,
following routes of
transmission similar to HIV-1 (Harrison, et al. ( 1991 ) Journal of Acquired
Immune
Deficiency Syndromes 4, 11 SS-60; Kanki, et al. ( 1992) American Journal of
Epidemiology
136, 895-907; Romieu, et al. ( 1990) Journal of Acquired Immune Deficiency
Syndromes 3,
220-30). Epidemiological studies suggest that HIV-2 produces human disease
with lesser
penetrance than HIV-1, and exhibits a considerably longer period of clinical
latency (at least
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
25 years, and possibly longer, as opposed to less than a decade for HIV-1;
see, Kanki, et al.
( 1991 ) Aids Clinical Review I 991, 17-38; Romieu, et al. ( 1990) Journal of
Acguired Immune
Deficiency Syndromes 3, 220-30, and Travers et al. ( 1995) Science 268: 1612-
1615).
The ability of HIV virus populations to rapidly point mutate to avoid the
immune response poses a special challenge for vaccine design. While the immune
system
has responded to viruses in a gradual and co-evolutionary manner, the present
invention
provides a general approach that provides for massively faster evolution to
produce new
vaccines to stimulate more effective immune responses.
For example, during the incubation period for HIV infection, which lasts for
several years, low titers of HIV can result from high HIV replication rates in
conjunction
with efficient viral clearance by the immune system. In response to these
selective forces,
virus mutations are selected which reduce recognition and neutralization by
the immune
system's B and T-cell responses. See, Lukashov et al. (1995) J. Virol. 69:6911-
6916. During
the long incubation time, these mutations accumulate and eventually overwhelm
the immune
system's defenses. See, Ho et al. ( 1995) Nature.
Live attenuated vaccines, typically produced by prolonged growth of human
viruses in animal cells, have proven useful as vaccines for several diseases,
including mumps,
rubella and measles. Attenuation involves the slow accumulation of many
mutations
throughout the viral genome during the course of adapting to growth in the
animal cells.
When used to vaccinate humans, the attenuated virus grows only weakly and
elicits a
complex immune response which the virus is unable to avoid. The mutations in
the
attenuated virus could, in principle, revert in the same stepwise fashion that
it underwent to
grow in culture.
The risk of reversion is highest in viruses with a high mutation rate such as
HIV-1, which makes this strategy dangerous under current techniques for
vaccine
development. It is worth noting, however, that protective effects against HIV-
1 are observed
following infection with the related HIV-2 virus, which is much less
pathogenic than HIV-1.
Thus, protective effects against HIV can be achieved with live vaccines.
To reduce the risk of reversion, a large number of mutations need to
accumulate in the virus. However, if too many mutations are present, the
immune system in
36
CA 02331335 2000-12-21
WO 00/18906 PC'TNS99/22588
effect recognizes the attenuated virus, but not the virus against which a
protective effect is
sought.
As provided herein, immunogenic compositions such as vaccines are created
which contain a large number of silent substitutions. In contrast to existing
attenuated
S viruses, such viruses have native protein sequences and elicit essentially
the same immune
responses as the corresponding wild-type virus (typically one or a few
additional disabling
mutations can also be incorporated). Colon alteration results in two effects
that both
increase the potential of the vaccine.
First, like standard attenuated viruses, the growth of colon-altered viruses
is
attenuated, due to the effect of the colon alterations on translation,
regulatory sequences,
mRNA folding, packaging, and the like. For example, regulation of HIV-1
envelope
expression has been observed as a result of colon usage. See, Haas et al. (
1989) Current
BioloQV 6(3):315-324.
Second, colon alteration results in impairment of virus evolution. As
1 S discussed above, modification of the colons alters the mutational escape
spectrum of the
virus, upsetting the evolutionary selection for specific colons.
The six colon amino acids axe the best targets for colon alteration. Serine,
arginine and leucine each have one group of four colons, plus two colons in an
unrelated
group. See, Figure 12. Switching all of the serine colons from AGY to TCX and
vice versa,
yields proteins with unaltered amino acid sequences. See also, Figure 13.
However, these
colon groups differ significantly in the spectrum of the amino acids that they
yield upon
point mutation. Of all possible point mutations of one colon for serine (TCA)
78% result in
a different amino acid compared to point mutations obtained for the AGT colon
for serine.
See, Figure 13. A virus with hundreds of colon alterations is in,
statistically, a very different
2S mutational space, able to access a totally different mutation spectrum, or
"cloud," compared
to the wild-type virus. The overall strategy for producing an evolution-
defective virus is
additionally set forth in Figures 14, 1 S and 17. Figure 16, panels A-C show
results of single
mutations of different colons for ser, arg, and ieu.
Point mutation is critical for viruses such as HIV-1 to stay ahead of the host
immune system. The amino acid mutations that are required for virus escape are
likely not
random. Wild type colon usage has evolved to allow optimal immune system
evasion. The
37
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
wild type codon usage is likely to favor mutations that represent alterations
that avoid the
host immune system, without detrimentally affecting the proteins) encoded.
While complex,
this natural pattern of amino acid sequence change of the natural virus in
response to the host
system is non-random and weakly predictable. See also, Seiller-Moiseiwitsch et
al. ( 1994)
Annu. Rev. Genet. 28:559-596.
Changing all of the codons for ser, arg and leu in the 875 as envelope
polyprotein of HIV-1 (e.g., strain MN) would affect 187 codons {22%) resulting
in 561
mutations. See, e.g., Figure 18, panels A-D. If alI of the HIV proteins were
altered, the
number of mutations would be more than three-fold higher. The construction of
such codon
modified viruses is simplified by recent advances in the synthesis of long DNA
sequences,
which enable the assembly of a plasmid of average size from 40 mer oligos in a
single step
with about 75% efficiency. See, Stemmer ( 1994) Nature 370 389-391. See also,
Figure 19
for a list of oligos in one application for synthesis of HIV Env. While the
synthesis of the
envelope gene is sufficient, synthesis of the whole HIV genome from oligos can
be
performed by this method.
In practice, a preferred balance of attenuation and evolution impairment is
obtained by DNA shuffling (e.g., Stemmer et al. (1995) Gene 164:49-53), e.g.,
of the wild-
type and codon altered sequences, followed by selection of the resulting
library of viruses
that retain moderate growth despite many codon alterations.
While attenuation that can be obtained by this approach may be sufficient for
obtaining a vaccine for most viruses, for HIV-1, the evolution impairment is
more important,
due to the high mutation rate of the virus. Live vaccines are used only if
they elicit an
immune response which is complex and strong enough to prevent infection of the
wild-type
virus. Live virus vaccines are typically more protective than single protein
vaccines because
it is harder to out-mutate T and B-cell responses to a larger number of
epitopes. The weak
growth of the live virus vaccine results in a larger antigenic dose and point
mutation is
increases the complexity of the immune response. To evaluate vaccine
competence, vaccine
potential is evaluated in Macaques (M. nemestrina) or chimpanzees using SIV
variants that
are known to cause AIDS. Sequence for an example SIV, SIVsmm, is found at Gene
Bank
Accession No. x 14307. This virus is closely related to HIV-2. See, Hirsch (
1989) Nature
339: 389-392. In general, many complete sequences for HIVs, SIVs and many
other viruses
38
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
are found in well known sequence repositories, including GenBank, EMBL, DDBJ
and the
NCBI. Well characterized HIV clones include: HIV-1NL43, HIV-1SF2, HIV-1BRU and
HIV-1 MN. For an introduction to the genetic variability of HIV, see, SeiIlier-
Moiseiwitsch
et al. (1994) Annu. Rev. Genet. 28:559-96 and the references cited therein.
Several HIV-2 isolates, including three molecular clones of HiV-2 (HIV-2RO~,
HIV-2ss~-isY, and HIV-2UC~), have also been reported to infect macaques (M.
mulatta and M.
nemestrina) or baboons (Franchini, et al. (1989) Proc. Natl. Acad. Sci. U.S.A.
86, 2433-2437;
Barnett, et al. ( 1993) Journal of Virology 67, 1006-14; Boeri, et al. ( 1992)
Journal of
Virology 66, 4546-50; Castro, et al. ( 1991 ) Virology 184, 219-26; Franchini,
et al. (1990)
Journal of Virology 64, 4462-7; Putkonen, et al. ( 1990) Aids 4, 783-9;
Putkonen, et al. ( 1991 )
Nature 352, 436-8). As human pathogens capable of infection of small primates,
HIV-2
molecular clones provide attractive models for studies of AIDS pathogenesis,
and for drug
and vaccine development against HIV-1 and HIV-2.
Recently, HIV-2 was suggested as a possible vaccine candidate against the
more virulent HIV-1 due to its long asymptomatic latency period, and its
ability to protect
against infection by HIV-1 (see, Travers et al. (1995) Science 268: 1612-1615
and related
commentary by Cohen et al (1995) Science 268: 1566). In the nine-year study by
Travers et
al. (id) of West African prostitutes infected with HIV-2, it was determined
that infection with
HIV-2 caused a 70% reduction in infection by HIV-1. Thus, codon altered HIV-2
viruses can
also be used as a live vaccine, against both HIV-2 and HIV-1. Furthermore,
because the
natural pathogenicity of HIV-2 is less than HIV-1, it is, in addition to HIV-
l, a preferred
virus for modification.
FORMATS FOR SEQUENCE RECOMBINATION
The methods of the invention entail performing recombination ("shuffling")
and screening or selection to "evolve" individual genes, whole plasmids or
viruses, multigene
clusters, or even whole genomes (Stemrner (1995) BiolTechnology 13:549-553).
Reiterative
cycles of recombination and screening/selection can be performed to further
evolve the
nucleic acids of interest. Such techniques do not require the extensive
analysis and
computation required by conventional methods for polypeptide engineering.
Shuffling
allows the recombination of large numbers of mutations in a minimum number of
selection
cycles, in contrast to natural pair-wise recombination events (e.g., as occur
during sexual
39
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
replication). Thus, the sequence recombination techniques described herein
provide
particular advantages in that they provide recombination between mutations in
any or all of
these, thereby providing a very fast way of exploring the manner in which
different
combinations of mutations can affect a desired result. In some instances,
however, structural
and/or functional information is available which, although not required for
sequence
recombination, provides opportunities for modification of the technique.
Exemplary formats and examples for sequence recombination, referred to,
e.g., as "DNA shuffling," "fast forced evolution," or "molecular breeding,"
have been
described by the present inventors and co-workers in the following patents and
patent
applications: US Patent No. 5,605,793; PCT Application WO 95/22625 (Serial No.
PCT/US95/02126), filed February 17, 1995; US Serial No. 08/425,684, filed
April 18, 1995;
US Serial No. 08/621,430, filed March 25, 1996; PCT Application WO 97/20078
(Serial No.
PCT/L1S96/05480), filed April 18, 1996; PCT Application WO 97/35966, filed
March 20,
1997; US Serial No. 08/675,502, filed July 3, 1996; US Serial No. 08/721, 824,
filed
September 27, 1996; PCT Application WO 98/13487, filed September 26, 1997;
"Evolution
of Whole Cells and Organisms by Recursive Sequence Recombination" Attorney
Docket No.
018097-020720US filed July 15, 1998 by del Cardayre et al. (PCT/LJS99/15972,
filed
07/15/1999); Stemmer, Science 270:1510 (1995); Stemmer et al., Gene 164:49-53
(1995);
Stemmer, BiolTechnology 13:549-553 (1995); Stemmer, Proc. Natl. Acad. Sci.
U.S.A.
91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994); Crameri et al.,
Nature
Medicine 2(1):1-3 (1996); and Crameri et al., Nature Biotechnology 14:315-319
(1996), each
of which is incorporated by reference in its entirety for all purposes.
The recombination procedure starts with at least two substrates that generally
show substantial sequence identity to each other (e.g., at least about 30%,
50%, 70%, 80% or
90% or more sequence identity), but differ from each other at certain
positions. For example,
at least one codon altered nucleic acid is recombined with one or more
additional nucleic acid
(the additional nucleic acid can also be a codon altered nucleic acid) herein.
The difference
between nucleic acids to be recombined can be any type of mutation, for
example,
substitutions, insertions and deletions. Often, different segments differ from
each other in
about S-20 positions. For recombination to generate increased diversity
relative to the
starting materials, the starting materials must differ from each other in at
least two nucleotide
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
positions. That is, if there are only two substrates, there should be at least
two divergent
positions. If there are three substrates, for example, one substrate can
differ from the second
at a single position, and the second can differ from the third at a different
single position.
The starting DNA segments can be natural variants of each other, for example,
allelic or
species variants. More typically, they will be codon altered nucleic acids
derived from one or
more homologous nucleic acid sequence. The segments can also be from
nonallelic genes
showing some degree of structural and usually functional relatedness (e.g.,
codon altered
nucleic acids derived from different, but homologous, genes within a
superfamily). The
starting DNA segments can also be induced variants of each other. For example,
one DNA
segment can be produced by error-prone PCR replication of the other, or by
substitution of a
mutagenic cassette. Induced mutants can also be prepared by propagating one
(or both) of
the segments in a mutagenic strain. In these situations, strictly speaking,
the second DNA
segment is not a single segment but a large family of related segments. The
different
segments forming the starting materials are often the same length or
substantially the same
1 S length. However, this need not be the case; for example; one segment can
be a subsequence
of another. The segments can be present as part of larger molecules, such as
vectors, or can
be in isolated form.
The starting DNA segments are recombined by any of the sequence
recombination formats provided herein to generate a diverse library of
recombinant DNA
segments. Such a library can vary widely in size from having fewer than 10 to
more than
1 O5, 1 O9, 1 O t'', 10 ~ 5, 1 Oz° or even more members. In some
embodiments, the starting
segments and the recombinant libraries generated will include essentially full-
length coding
sequences and any essential regulatory sequences, such as a promoter and
polyadenylation
sequence, required fox expression. In other embodiments, the recombinant DNA
segments in
the library can be inserted into a common vector providing sequences necessary
for
expression before performing screening/selection.
Use of RestrictionEnzyme Sites to Recombine Mutations
In some situations it is advantageous to use restriction enzyme sites in
nucleic
acids to direct the recombination of mutations in a nucleic acid sequence of
interest. These
techniques are particularly preferred in the evolution of fragments that
cannot readily be
shuffled by other existing methods due to the presence of repeated DNA or
other problematic
41
CA 02331335 2000-12-21
WO 00/18906 PCTNS99/22588
primary sequence motifs. These situations also include recombination formats
in which it is
preferred to retain certain sequences unmutated. The use of restriction enzyme
sites is also
preferred for shuffling large fragments (typically greater than 10 kb), such
as gene clusters
that cannot be readily shuffled and "PCR-amplified" because of their size.
Although
fragments up to 50 kb have been reported to be amplified by PCR (Barnes, Proc.
Natl. Acad.
Sci. U.S.A. 91:2216-2220 (1994)), it can be problematic for fragments over 10
kb, and thus
alternative methods for shuffling in the range of 10 - 50 kb and beyond are
preferred.
Preferably, the restriction endonucleases used are of the Class II type
(Sambrook, Ausubel
and Berger, supra) and of these, preferably those which generate
nonpalindromic sticky end
overhangs such as Alwn I, Sfi I or BstXl. These enzymes generate
nonpalindromic ends that
allow for efficient ordered reassembly with DNA ligase. Typically, restriction
enzyme (or
endonuclease) sites are identified by conventional restriction enzyme mapping
techniques
(Sambrook, Ausubel, and Berger, szrpra.), by analysis of sequence information
for that gene,
or by introduction of desired restriction sites into a nucleic acid sequence
by synthesis (i.e. by
incorporation of silent mutations). For example, one or more codon-altered
nucleic acid can
be recombined at restriction sites, e.g., with one or more nucleic acid of
interest (including,
e.g. a gene or gene cluster to be modified by recombination with the codon-
altered nucleic
acid).
The DNA substrate molecules to be digested can either be from in vivo
replicated DNA, such as a plasmid preparation, or from synthetic or e.g., PCR
amplified
nucleic acid fragments harboring the restriction enzyme recognition sites of
interest,
preferably near the ends of the fragment. Typically, at least two variants of
a gene of interest,
each having one or more mutations, and at least one of which incorporating
codon-
modifications, are digested with at least one restriction enzyme determined to
cut within the
nucleic acid sequence of interest. The restriction fragments are then joined
with DNA ligase
to generate full length genes having shuffled regions. The number of regions
shuffled will
depend on the number of cuts within the nucleic acid sequence of interest. The
shuffled
molecules can be introduced into cells as described above and screened or
selected for a
desired property as described herein. Nucleic acid can then be isolated from
pools (libraries),
or clones having desired properties and subjected to the same procedure until
a desired
degree of improvement is obtained.
42
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
In some embodiments, at least one DNA substrate molecule or fragment
thereof is isolated and subjected to mutagenesis. In some embodiments, the
pool or library of
religated restriction fragments are subjected to mutagenesis or additional
recombination
protocols before the digestion-ligation process is repeated. "Mutagenesis" as
used herein
comprises such techniques known in the art as PCR mutagenesis, oligonucleotide-
directed
mutagenesis, site-directed mutagenesis, etc., and recursive sequence
recombination by any of
the techniques described herein.
Reassembly PCR
A further technique for recombining mutations in a nucleic acid sequence
utilizes "reassembly PCR." This method can be used to assemble multiple
segments that
have been separately evolved into a full length nucleic acid template such as
a gene. This
technique is performed when a pool of advantageous mutants is known from
previous work
or has been identified by screening mutants that may have been created by any
mutagenesis
technique known in the art, such as PCR mutagenesis, cassette mutagenesis,
doped oligo
mutagenesis, chemical mutagenesis, or propagation of the DNA template in vivo
in mutator
strains. Boundaries defining segments of a nucleic acid sequence of interest
preferably lie in
intergenic regions, introns, or areas of a gene not likely to have mutations
of interest.
Preferably, oligonucleotide primers (oligos) are synthesized for PCR
amplification of
segments of the nucleic acid sequence of interest, such that the sequences of
the
oligonucleotides overlap the junctions of two segments. The overlap region is
typically about
10 to 100 nucleotides in length.
Each of the segments is amplified with a set of such primers. The PCR
products are then "reassembled" according to assembly protocols such as those
discussed
herein to assemble randomly fragmented genes. In brief, in an assembly
protocol the PCR
products are first purified away from the primers, by, for example, gel
electrophoresis or size
exclusion chromatography. Purified products are mixed together and subjected
to about 1-10
cycles of denaturing, reannealing, and extension in the presence of polymerase
and
deoxynucleoside triphosphates (dNTPs) and appropriate buffer salts in the
absence of
additional primers ("self priming"). Subsequent PCR with primers flanking the
gene are used
to amplify the yield of the fully reassembled and shuffled genes. In some
embodiments, the
resulting reassembled genes are subjected to mutagenesis before the process is
repeated.
43
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
In the present invention, oligos such as PCR primers can include codon
modifications as compared to a starting sequence. In addition,
oligonucleotides can form the
basis for PCR concatemerization reactions in which overlapping hybridized
oligonucleotides
are extended in one or more PCR amplification cycles. In this embodiment, a
template
S nucleic acid is not required (although a template or fragments thereof can
be added to the
amplification mixture, which can aid in the eventual reassembly of a full-
length gene).
Further details regarding oligonucleotide gene reassembly methods are found,
e.g., in
Crameri et al. "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION"
filed February 5, 1999, USSN 60/118,813 and Crameri et al. "OLIGONUCLEOTIDE
MEDIATED NUCLEIC ACID RECOMBINATION" filed June 24, 1999, USSN 60/141,049.
In a further embodiment, the PCR primers for amplification of segments of a
nucleic acid sequence of interest are used to introduce variation into the
gene of interest as
follows. Mutations at sites of interest in a nucleic acid sequence are
identified by screening
or selection, by sequencing homologues of the nucleic acid sequence, and so
on.
Oligonucleotide PCR primers are synthesized which encode wild type or mutant
information
at sites of interest. These primers are then used in PCR mutagenesis to
generate libraries of
full length genes encoding permutations of wild type and mutant information at
the
designated positions. This technique is typically advantageous in cases where
the screening
or selection process is expensive, cumbersome, or impractical relative to the
cost of
sequencing the genes of mutants of interest and synthesizing mutagenic
oligonucleotides.
Site Directed Muta~~enesis (SDM) with Oligonucleotides Encoding
I-iomolo~ue Mutations Followed by Shuffling
In some embodiments of the invention, sequence information from one or
more substrate sequences is added to a given "parental" sequence of interest,
with subsequent
recombination between rounds of screening or selection. Typically, this is
done with site-
directed mutagenesis performed by techniques well known in the art (e.g.,
Bergen Ausubel
and Sambrook, supra.) with one substrate as a template and oligonucleotides
encoding single
or multiple mutations from other substrate sequences, e.g. homologous genes.
After
screening or selection for an improved phenotype of interest, the selected
recombinants) can
be further evolved using recursive techniques. After screening or selection,
site-directed
mutagenesis can be done again with another collection of oligonucleotides
encoding
44
CA 02331335 2000-12-21
WO 00/18906 PCTNS99/22588
homologue mutations, and the above process repeated until the desired
properties are
obtained.
When the difference between two homologues is one or more single point
mutations in a codon, degenerate oligonucleotides can be used that encode the
sequences in
both homologues. One oligonucleotide can include many such degenerate codons
and still
allow one to exhaustively search all permutations over that block of sequence.
When the homologue sequence space is very large, it can be advantageous to
restrict the search to certain variants. Thus, for example, computer modeling
toots (Lathrop
et al. ( 1996) J. Mol. Biol., 255: 641-665) can be used to model each
homologue mutation
onto the target protein and discard any mutations that are predicted to
grossly disrupt
structure and function. In silico genetic algorithm operations for generating
and predicting
mutational events are found in Selifonov and Stemmer "METHODS FOR MAKING
CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED
CHARACTERISTICS" filed 02/05/1999, USSN 60/118854.
Oli;~onucieotide and in silico shuffling formats
As mentioned above, at least two additional related formats are useful in the
practice of the present invention. The first, referred to as "in silico"
shuffling utilizes
computer algorithms to perform "virtual" shuffling using genetic operators in
a computer. As
applied to the present invention, codon altered gene sequence strings are
recombined in a
computer system and desirable products are made, e.g., by reassembly PCR or
ligation of
synthetic oligonucleotides. In silico shuffling is described in detail in
Selifonov and Stemmer
in "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES &
POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" filed 02/05/1999, USSN
60/118854. In brief, genetic operators (algorithms which represent given
genetic events such
as point mutations, recombination of two strands of homologous nucleic acids,
etc.) are used
to model recombinational or mutational events which can occur in one or more
nucleic acid,
e.g., by aligning nucleic acid sequence strings (using standard alignment
software, or by
manual inspection and alignment) and predicting recombinational outcomes. The
predicted
recombinational outcomes are used to produce corresponding molecules, e.g., by
oligonucleotide synthesis and reassembly PCR.
CA 02331335 2000-12-21
WO 00/18906 PCTNS99/22588
The second useful format is referred to as "oligonucleotide mediated
shuffling" in which oligonucleotides corresponding to a family of related
homologous nucleic
acids (e.g., as applied to the present invention, colon modified synthetic
homologous variants
of a nucleic acid) which are recombined to produce selectable nucleic acids.
This format is
S described in detail in Crameri et al. "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID
RECOMBINATION" filed February 5, 1999, USSN 60/118,813 and Crameri et al.
"OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" filed June 24,
1999, USSN 60/141,049. In brief, selected oligonucleotides are synthesized,
ligated and
elongated, typically either in a polymerise or ligase-mediated elongation
reaction. The
technique can be used to recombine homologous or even non-homologous codon-
altered
nucleic acid sequences.
One advantage of oligonucleotide-mediated recombination is the ability to
recombine homologous nucleic acids with low sequence similarity, or even non-
homologous
nucleic acids. In these low-homology oligonucieotide shuffling methods, one or
more set of
1 S fragmented nucleic acids (e.g., cleaved codon-modified oligonucleotides,
or synthesized
codon-modified oligonucleotides) are recombined, e.g., with a with a set of
crossover family
diversity oligonucleotides. Each of these crossover oligonucleotides have a
plurality of
sequence diversity domains corresponding to a plurality of sequence diversity
domains from
homologous or non-homologous nucleic acids with low sequence similarity. The
fragmented
oligonucleotides, which are derived by comparison to one or more homologous or
non-
homologous nucleic acids, can hybridize to one or more region of the crossover
oligos,
facilitating recombination.
When recombining homologous nucleic acids, sets of overlapping family gene
shuffling oligonucleotides (which are derived by comparison of homologous
nucleic acids
that include one or more codon-modified nucleic acid, followed by synthesis of
corresponding oligonucleotides) are hybridized and elongated (e.g., by
reassembly PCR or
ligation), providing a population of recombined nucleic acids, which can be
selected for a
desired trait or property. The set of overlapping family shuffling gene
oligonucleotides
includes a plurality of oligonucleotide member types which have consensus
region
subsequences derived from a plurality of homologous target nucleic acids.
46
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
Typically, as applied to the present invention, family gene shuffling
oligonucleotide which include one or more codon-altered nucleic acids) are
provided by
aligning homologous nucleic acid sequences to select conserved regions of
sequence identity
and regions of sequence diversity. A plurality of family gene shuffling
oligonucleotides are
S synthesized (serially or in parallel) which correspond to at least one
region of sequence
diversity.
Sets of fragments, or subsets of fragments used in oligonucleotide shuffling
approaches can be provided by cleaving one or more homologous nucleic acids
(e.g., with a
DNase), or, more commonly, by synthesizing a set of oligonucleotides
corresponding to a
plurality of regions of at least one nucleic acid (typically oligonucleotides
corresponding to a
full-length nucleic acid are provided as members of a set of nucleic acid
fragments). In the
shuffling procedures herein, these cleavage fragments can be used in
conjunction with family
gene shuffling oligonucleotides, e.g., in one or more recombination reaction
to produce
recombinant codon-altered nucleic acid(s).
Additional oligonucleotide shuffling formats are found in co-filed application
by Crameri et al., "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID
RECOMBINATION" {Attorney Docket Number 02-296-2US) and in co-filed application
by
Welch et al., "USE OF CODON VARIED OLIGONUCLEOTIDE SYNTHESIS FOR
SYNTHETIC SHUFFLING" (Attorney docket number 02-1007). In particular, these
applications provide for tri-nucleotide-based synthesis of degenerate
oligonucleotides,
thereby providing for codon substitution during oligonucleotide shuffling. In
brief, this
procedure utilizes tri-nucleotide phosphoramidite chemistry to synthesize
oligos, rather than
standard mono-nucleotide synthesis. Because codons are altered as a unit, the
synthetic
scheme of degenerate oligonucleotides is simplified.
Additional In Vitro DNA Shuffling Formats
In one embodiment for shuffling DNA sequences in vitro, the initial substrates
for recombination are a pool of related sequences, e.g., different variant
forms, as homologs
from different individuals, strains, or species of an organism, or related
sequences from the
same organism, as allelic variations. The sequences can be DNA or RNA and can
be of
various lengths depending on the size of the gene or DNA fragment to be
recombined or
reassembled. Preferably the sequences are from 50 base pairs (bp) to 50
kilobases (kb).
47
CA 02331335 2000-12-21
WO 00/18906 PCT/US99I22588
The pool of related substrates are converted into overlapping fragments, e.g.,
from about 5 by to 5 kb or more. Often, for example, the size of the fragments
is from about
by to 1000 bp, and sometimes the size of the DNA fragments is from about 100
by to 500
bp. The conversion can be effected by a number of different methods, such as
DNase I or
5 RNase digestion, random shearing or partial restriction enzyme digestion, or
by
oligonucleotide synthesis as in the family oligonucleotide-mediated shuffling
methods of
crameri et al., discusses supra. For discussions of protocols for the
isolation, manipulation,
enzymatic digestion, and the like, of nucleic acids, see, for example,
Sambrook et al. and
Ausubel, both supra. The concentration of nucleic acid fragments of a
particular length and
10 sequence is often less than 0.1 % or 1 % by weight of the total nucleic
acid. The number of
different specific nucleic acid fragments in the mixture is usually at least
about 2, 10, 100,
500 or 1,000 or more.
The mixed population of nucleic acid fragments are converted to at least
partially single-stranded form using any of a variety of techniques,
including, for example,
heating, chemical denaturation, use of DNA binding proteins, and the like (in
oligonucleotide
mediated methods, this step can be omitted). Conversion can be effected by
heating to about
80 °C to 100 °C, more preferably from 90 °C to 96
°C, to form single-stranded nucleic acid
fragments and then reannealing. Conversion can also be effected by treatment
with a single-
stranded DNA binding protein (see Wold (1997) Annu. Rev. Biochem. 66:61-92) or
recA
protein (see, e.g., Kiianitsa ( 1997) Proc. Natl. Acad. Sci. U S A 94:7837-
7840). Single-
stranded nucleic acid fragments having regions of sequence identity with other
single-
stranded nucleic acid fragments can then be reannealed by cooling to 20
°C to 75 °C, and
preferably from 40 °C to 65 °C. Renaturation can be accelerated
by the addition of
polyethylene glycol (PEG), other volume-excluding reagents or salt. The salt
concentration
is preferably from 0 mM to 200 mM, more preferably the salt concentration is
from 10 mM to
100 mM. The salt may be KCl or NaCI. The concentration of PEG is preferably
from 0% to
20%, more preferably from 5% to 10%. The fragments that reanneal can be from
different
substrates. The annealed nucleic acid fragments are incubated in the presence
of a nucleic
acid polymerase, such as Taq or Klenow, and dNTP's (i.e. dATP, dCTP, dGTP and
dTTP). If
regions of sequence identity are large, Taq polymerase can be used with an
annealing
temperature of between 45-65 °C. If the areas of identity are small,
Klenow polymerase can
48
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
be used with an annealing temperature of between 20-30 °C. The
polymerase can be added
to the random nucleic acid fragments prior to annealing, simultaneously with
annealing or
after annealing.
The process of denaturation, renaturation and incubation in the presence of
polymerase or ligase of overlapping fragments to generate a collection of
polynucleotides
containing different permutations of fragments is sometimes referred to as
shuffling of the
nucleic acid in vitro. This cycle is repeated for a desired number of times.
Preferably the
cycle is repeated from 2 to 100 times, more preferably the sequence is
repeated from 10 to 40
times. The resulting nucleic acids are a family of double-stranded
polynucleotides of from
about 50 by to about 100 kb, preferably from 500 by to SO kb. The population
represents
variants of the starting substrates showing substantial sequence identity
thereto but also
diverging at several positions. The population has many more members than the
starting
substrates. The population of fragments resulting from shuffling is used to
transform host
cells, optionally after cloning into a vector.
In one embodiment utilizing in vitro shuffling, subsequences of recombination
substrates can be generated by amplifying the full-length sequences under
conditions which
produce a substantial fraction, typically at least 20 percent or more, of
incompletely extended
amplification products. Another embodiment uses random primers to prime an
entire
template DNA to generate less than full length amplification products. The
amplification
products, including the incompletely extended amplification products are
denatured and
subjected to at least one additional cycle of reannealing and amplification.
This variation, in
which at least one cycle of reannealing and amplification provides a
substantial fraction of
incompletely extended products, is termed "stuttering." In the subsequent
amplification
round, the partially extended (less than full length) products reanneal to and
prime extension
on different sequence-related template species. In another embodiment, the
conversion of
substrates to fragments can be effected by partial PCR amplification of
substrates.
In another embodiment, a mixture of fragments is spiked with one or more
oligonucleotides. The oligonucleotides can be designed to include
precharacterized
mutations of a wildtype sequence (e.g., codon modification), or sites of
natural variations
between individuals or species. The oligonucleotides also typically include
sufficient
sequence or structural homology flanking such mutations or variations to allow
annealing
49
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
with the wildtype fragments. Annealing temperatures can be adjusted depending
on the
length of homology.
In a further embodiment, recombination occurs in at least one cycle by
template switching, such as when a DNA fragment derived from one template
primes on the
homologous position of a related but different template. Template switching
can be induced
by addition of recA (see, Kiianitsa ( 1997) supra), rad51 (see, Namsaraev (
1997) Mol. Cell.
Biol. 17:5359-5368), rad55 (see, Clever ( 1997) EMBO J. 16:2535-2544), rad57
(see, Sung
(1997} Genes Dev. 11:1111-1121) or other polymerases {e.g., viral poiymerases,
reverse
transcriptase) to the amplification mixture. Template switching can also be
increased by
increasing the DNA template concentration.
Another embodiment utilizes at least one cycle of amplification, which can be
conducted using a collection of overlapping single-stranded DNA fragments of
related
sequence, and different lengths. Fragments can be prepared using a single
stranded DNA
phage, such as M13 (see, Wang (1997) Biochemistry 36:9486-9492). Each fragment
can
hybridize to and prime polynucleotide chain extension of a second fragment
from the
collection, thus forming sequence-recombined polynucleotides. In a further
variation,
ssDNA fragments of variable length can be generated from a single primer by
Pfu, Taq, Vent,
Deep Vent, UlTma DNA polymerase or other DNA polymerases on a first DNA
template
{see, Cline (1996) Nucleic Acids Res. 24:3546-3551). The single stranded DNA
fragments
are used as primers for a second, Kunkel-type template, consisting of a uracil-
containing
circular ssDNA. This results in multiple substitutions of the first template
into the second.
See, Levichkin (1995) Mol. Biology 29:572-577; Jung (1992) Gene 121:17-24.
In some embodiments of the invention, shuffled nucleic acids obtained by use
of the recursive recombination methods of the invention, are put into a cell
and/or organism
for screening. Shuffled genes can be introduced into, for example, bacterial
cells, yeast cells,
fungal cells vertebrate cells, invertebrate cells or plant cells for initial
screening. Bacillus
species (such as B. subtilis and E. coli are two examples of suitable
bacterial cells into which
one can insert and express shuffled genes which provide for convenient
shuttling to other cell
types (a variety of vectors for shuttling material between these bacterial
cells and eukaryotic
cells are available; see, Sambrook, Ausubel and Berger, all supra). The
shuffled genes can
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
be introduced into bacterial, fungal or yeast cells either by integration into
the chromosomal
DNA or as plasmids.
Bacterial, plant, animal and yeast systems are preferred in the present
invention. For example, in one embodiment, shuffled genes can be introduced
into plant or
animal cells for production purposes (it will be appreciated that transgenic
plants are,
increasingly, an important source of industrial enzymes), or can be introduced
into a plant or
animal cell for therapeutic purposes. Thus, a transgene of interest can be
modified using the
recursive sequence recombination methods of the invention in vitro and
reinserted into the
cell for in vivolin situ selection for the new or improved property, in
bacteria, eukaryotic
cells, or whole eukaryotic organisms.
In Vivo DNA Shuffling Formats
In some embodiments of the invention, DNA substrate molecules, e.g., those
comprising codon modifications relative to a wild-type sequence, are
introduced into cells,
where the cellular machinery directs their recombination. For example, a
library of mutants
is constructed and screened or selected for mutants with improved phenotypes
by any of the
techniques described herein.
The DNA substrate molecules encoding the best candidates are recovered by
any of the techniques described herein, then fragmented and used to transfect
a plant host and
screened or selected for improved function. If further improvement is desired,
the DNA
substrate molecules are recovered from the host cell, such as by PCR, and the
process is
repeated until a desired level of improvement is obtained. In some
embodiments, the
fragments are denatured and reannealed prior to transfection, coated with
recombination
stimulating proteins such as recA, or co-transfected with a selectable marker
such as Neon to
allow the positive selection for cells receiving recombined versions of the
gene of interest.
Methods for in vivo shuffling are described in, for example, PCT application
WO 98/13487
and WO 97/20078. The efficiency of in vivo shuffling can be enhanced by
increasing the
copy number of a gene of interest in the host cells.
Whole Genome Shuffling
In one embodiment, the selection methods herein are utilized in a "whole
genorne shuffling" format. An extensive guide to the many forms of whole
genome shuffling
is found in the pioneering application to the inventors and their co-workers
entitled
51
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
"Evolution of Whole Cells and Organisms by Recursive Sequence Recombination,"
PCT/tTS99/15972, by del Cardayre et al. Any codon-altered set ofnucleic acids
can be used
to transform cells, which can then be shuffled by in a whole genome format.
In brief, whole genome shuffling makes no presuppositions at all regarding
what nucleic acids may confer a desired property. Instead, entire genomes
(e.g., from a
genomic -library, or isolated from an organism) are shuffled in cells and
selection protocols
applied to the cells. These genomes can be spiked with any desired set of
nucleic acids,
including codon-modified nucleic acids.
Assays
The relevant assay for selection of a desired property of a codon-modified
nucleic acid will depend on the application. Many assays which detect activity
for proteins,
receptors, ligands, cells and the like are known. Formats include binding to
immobilized
components, cell or organismal viability, production of reporter compositions,
and the like.
In the high throughput assays of the invention, it is possible to screen up to
several thousand different shuffled variants in a single day. In particular,
each well of a
microtiter plate can be used to run a separate assay, or, if concentration or
incubation time
effects are to be observed, every 5-10 wells can test a single variant. Thus,
a single standard
microtiter plate can assay about 100 (e.g., 96) reactions. If 1536 well plates
are used, then a
single plate can easily assay from about 100- about 1500 different reactions.
It is possible to
assay several different plates per day; assay screens for up to about 6,000-
20,000 different
assays (i.e., involving different nucleic acids, encoded proteins,
concentrations, etc.) is
possible using the integrated systems of the invention. More recently,
microfluidic
approaches to reagent manipulation have been developed, e.g., by Caliper
Technologies
(Mountain View, CA).
In one aspect, library members, e.g., cells, viral plaques, spores or the
like, are
separated on solid media to produce individual colonies (or plaques). Using an
automated
colony picker (e.g., the Q-bot, Genetix, U.K.), colonies or plaques are
identified, picked, and
up to 10,000 different mutants inoculated into 96 well microtitre dishes
containing two 3 mm
glass balls/well. The Q-bot does not pick an entire colony but rather inserts
a pin through the
center of the colony and exits with a small sampling of cells, (or mycelia)
and spores (or
viruses in plaque applications). The time the pin is in the colony, the number
of dips to
52
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
inoculate the culture medium, and the time the pin is in that medium each
effect inoculum
size, and each can be controlled and optimized. The uniform process of the Q-
bot decreases
human handling error and increases the rate of establishing cultures (roughly
10,000/4 hours).
These cultures are then shaken in a temperature and humidity controlled
incubator. The
S glass balls in the microtiter plates act to promote uniform aeration of
cells and the dispersal
of mycelial fragments similar to the blades of a fermenter. Clones from
cultures of interest
can be cloned by limiting dilution. As also described supra, plaques or cells
constituting
libraries can also be screened directly for production of proteins, either by
detecting
hybridization, protein activity, protein binding to antibodies, or the like.
The ability to detect a subtle increase in the performance of a shuffled
library
member over that of a parent strain relies on the sensitivity of the assay.
The chance of
finding the organisms having an improvement is increased by the number of
individual
mutants that can be screened by the assay. To increase the chances of
identifying a pool of
sufficient size, a prescreen that increases the number of mutants processed
by, e.g., 10-fold
can be used. The goal of the primary screen is to quickly identify mutants
having equal or
better product titres than the parent strains) and to move only these mutants
forward to liquid
cell culture for subsequent analysis.
A number of well known robotic systems have also been developed for
solution phase chemistries useful in assay systems. These systems include
automated
workstations like the automated synthesis apparatus developed by Takeda
Chemical
Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic
arms (Zymate II,
Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto,
Calif.) which
mimic the manual synthetic operations performed by a scientist. Any of the
above devices
are suitable for use with the present invention, e.g., for high-throughput
screening of
molecules encoded by codon-altered nucleic acids. The nature and
implementation of
modifications to these devices (if any) so that they can operate as discussed
herein with
reference to the integrated system will be apparent to persons skilled in the
relevant art.
High throughput screening systems are commercially available (see, e.g.,
Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman
Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc. ).
These systems
typically automate entire procedures including all sample and reagent
pipetting, liquid
53
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
dispensing, timed incubations, and final readings of the microplate in
detectors) appropriate
for the assay. These configurable systems provide high throughput and rapid
start up as well
as a high degree of flexibility and customization.
The manufacturers of such systems provide detailed protocols the various high
throughput. Thus, for example, Zymark Corp. provides technical bulletins
describing
screening systems for detecting the modulation of gene transcription, ligand
binding, and the
like. Microfluidic approaches to reagent manipulation have also been
developed, e.g., by
Caliper Technologies (Mountain View, CA).
Optical images viewed (and, optionally, recorded) by a camera or other
recording device (e.g., a photodiode and data storage device) are optionally
further processed
in any of the embodiments herein, e.g., by digitizing the image and/or storing
and analyzing
the image on a computer. A variety of commercially available peripheral
equipment and
software is available for digitizing, storing and analyzing a digitized video
or digitized
optical image, e.g., using PC (Intel x86 or pentium chip- compatible DOSTM,
OS2TM
WINDOWSTM, WINDOWS NTTM or WINDOWS95TM based machines), MACINTOSHTM,
or UNIX based (e.g., SUNTM work station) computers.
One conventional system carries light from the assay device to a cooled
charge-coupled device (CCD) camera, in common use in the art. A CCD camera
includes an
array of picture elements (pixels). The light from the specimen is imaged on
the CCD.
Particular pixels corresponding to regions of the specimen (e.g., individual
hybridization sites
on an array of biological polymers) are sampled to obtain light intensity
readings for each
position. Multiple pixels are processed in parallel to increase speed. The
apparatus and
methods of the invention are easily used for viewing any sample, e.g., by
fluorescent or dark
field microscopic techniques.
Software elements for manipulating strings of characters which correspond to
codon-modified nucleic acids can be used to direct synthesis of
oligonucleotides relevant to
shuffling of codon-modified nucleic acids. Integrated systems comprising these
and other
useful features, e.g., a digital computer with additional features such as
high-throughput
liquid control software, image analysis software, data interpretation
software, a robotic liquid
control armature for transferring solutions from a source to a destination
operably linked to
the digital computer, an input device (e.g., a computer keyboard) for entering
data to the
54
CA 02331335 2000-12-21
WO 00/i8906 PCT/US99/22588
digital computer to control high throughput liquid transfer by the robotic
liquid control
armature an image scanner for digitizing label signals from labeled assay
components, or the
like are a feature of the invention.
In one aspect, the invention provides an integrated system comprising a
computer or computer readable medium comprising a database having at least two
artificial
homologous codon-altered nucleic acid sequence strings, and a user interface
allowing a user
to selectively view one or more sequence strings in the database. As discussed
theroughout,
there are a variety of sequence database programs for aligning and
manipulating sequences.
In addition, standard text manipulation software such as word processing
software (e.g.,
Microsft WordTM or Corel WodperfectTM) and database software (e.g.,
spreadsheet software
such as Microsoft ExcelTM, Corel Quattro ProTM, or database programs such as
Microsoft
AccessTM or ParadoxTM) can be used in conjuction with a user interface (e.g.,
a GUI in a
standard operating system such as a Windows, Macintosh or LINUX system) to
manipulate
strings of characters. Specialized alignment programs such as BLAST can also
be
I S incorporated into the systems of the invention for alignment of codon-
altered nucleic acids
(or corresponding character strings).
In addition to the integrated system elements mentioned above, the integrated
system can also include an automated oligonucleotide synthesizer operably
linked to the
computer or computer readable medium. Typically, the synthesizer is programmed
to
synthesize one or more oligonucleotide comprising one or more subsequence of
one or more
of the at least two artificial homologous codon-altered nucleic acids.
Modifications can be made to the method and materials as hereinbefore
described without departing from the spirit or scope of the invention as
claimed, and the
invention can be put to a number of different uses, including:
The use of an integrated system to test shuffled codon-modified DNAs,
including in an iterative process.
An assay, kit or system utilizing a use of any one of the selection
strategies,
materials, components, methods or substrates hereinbefore described. Kits will
optionally
additionally comprise instructions for performing methods or assays, packaging
materials,
one or more containers which contain assay, device or system components, or
the like.
CA 02331335 2000-12-21
WO 00/18906 PCT/US99/22588
In an additional aspect, the present invention provides kits embodying the
methods and apparatus herein. Kits of the invention optionally comprise one or
more of the
following: ( 1 ) a shuffled codon-modified component as described herein; (2)
instructions for
practicing the methods described herein, and/or for operating the selection
procedure herein;
(3) one or more assay component; (4) a container for holding nucleic acids or
enzymes, other
nucleic acids, transgneic plants, animals, cells, or the Like, (S) packaging
materials and (6)
software fixerd in a computer readable medium comprising sequences
corresponding to one
or more codon-altered nucleic acid character string.
In a further aspect, the present invention provides for the use of any
component or kit herein, for the practice of any method or assay herein,
and/or for the use of
any apparatus or kit to practice any assay or method herein.
While the foregoing invention has been described in some detail for purposes
of clarity and understanding, it will be clear to one skilled in the art from
a reading of this
disclosure that various changes in form and detail can be made without
departing from the
true scope of the invention. For example, all the techniques and materials
described above
can be used in various combinations. All publications and patent documents
cited in this
application are incorporated by reference in their entirety for all purposes
to the same extent
as if each individual publication or patent document were so individually
denoted.
56
