U.S. patent application number 14/792457 was filed with the patent office on 2016-03-24 for methods and compositions for targeted gene modification.
This patent application is currently assigned to Recombinetics, Inc.. The applicant listed for this patent is Recombinetics, Inc.. Invention is credited to Jeffrey J. Essner, Scott C. Fahrenkrug, Hsin-Kai Liao.
Application Number | 20160083746 14/792457 |
Document ID | / |
Family ID | 43544899 |
Filed Date | 2016-03-24 |
United States Patent
Application |
20160083746 |
Kind Code |
A1 |
Essner; Jeffrey J. ; et
al. |
March 24, 2016 |
METHODS AND COMPOSITIONS FOR TARGETED GENE MODIFICATION
Abstract
Disclosed herein are methods and compositions for gene targeting
utilizing fusion molecules comprising a recombinase domain and a
sequence-specific DNA-binding domain.
Inventors: |
Essner; Jeffrey J.; (Ames,
IA) ; Liao; Hsin-Kai; (San Diego, CA) ;
Fahrenkrug; Scott C.; (Minneapolis, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Recombinetics, Inc. |
Saint Paul |
MN |
US |
|
|
Assignee: |
Recombinetics, Inc.
|
Family ID: |
43544899 |
Appl. No.: |
14/792457 |
Filed: |
July 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12869232 |
Aug 26, 2010 |
9074224 |
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14792457 |
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PCT/US2010/044236 |
Aug 3, 2010 |
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12869232 |
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61230784 |
Aug 3, 2009 |
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Current U.S.
Class: |
435/462 |
Current CPC
Class: |
A61K 48/00 20130101;
C07K 2319/09 20130101; C12N 15/85 20130101; C07K 14/00 20130101;
A61P 43/00 20180101; C12N 2770/14022 20130101; C12N 15/90 20130101;
C12N 2800/30 20130101; C07K 2319/81 20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85 |
Goverment Interests
STATEMENT OF GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with government support under 1R21
RR025915-01 awarded by National Institute of Health. The Government
has certain rights in the invention.
Claims
1. A method of transfecting a cell comprising introducing into the
cell: an exogenous nucleic acid and a nucleoprotein filament of a
proteinaceous fusion molecule and a nucleic acid probe
complementary to a target site of DNA of the cell, wherein the
fusion protein comprises a recombinase domain that contributes to
the filament, a DNA-binding domain, and a nuclear localization
signal domain, wherein the exogenous nucleic acid is incorporated
into the DNA of the cell and expressed by the cell.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 12/869,232 filed Aug. 26, 2010, which is a continuation of
international application PCT/US2010/004236, filed on Aug. 3, 2010,
which claims priority to U.S. Provisional No. 61/230,784 filed Aug.
3, 2009 which are hereby incorporated by reference herein.
TECHNICAL FIELD
[0003] The technical field relates to targeted genome modification
including, but not limited to, targeted insertion, targeted
deletion, targeted gene inactivation and targeted mutagenesis.
BACKGROUND
[0004] A major area of interest in biology and medicine is the
targeted alteration of genomic nucleotide sequences. Such
alterations include insertion, deletion and replacement of
endogenous chromosomal nucleic acid sequences.
SUMMARY
[0005] Compositions and methods that will provide targeted
alteration of genomic sequences are disclosed. Certain fusion
proteins that safely and efficiently deliver exogenous sequences to
the intended site to achieve the desired effect are described and
also exemplified with working examples.
[0006] Past attempts have been made by others to alter genomic
sequences in cultured cells by taking advantage of the natural
phenomenon of homologous recombination. See, for example, Capecchi
(1989) Science 244:1288-1292; U.S. Pat. Nos. 6,528,313 and
6,528,314. If an exogenous polynucleotide has sufficient homology
to the genomic region containing the sequence to be altered, it is
possible for part or all of the sequence of the exogenous
polynucleotide to replace the genomic sequence by homologous
recombination. However, the frequency of homologous recombination
under these circumstances is extremely low. Moreover, the frequency
of insertion of the exogenous polynucleotide at genomic locations
that lack sequence homology exceeds the frequency of homologous
recombination by several orders of magnitude.
[0007] Thus, previous attempts to replace particular sequences have
involved contacting a cell ex vivo with an exogenous polynucleotide
(also referred to as donor DNA) comprising sequences bearing
homology to a targeted chromosomal region), followed by selection
of cells ex vivo in which the donor DNA molecule had undergone
homologous recombination into the genome. The success rate of these
methods is low, due to poor efficiency of homologous recombination
and a high frequency of non-specific insertion of the donor DNA
into regions of the genome other than the target site.
[0008] Because of these known problems with both the efficiency and
specificity of existing methods for targeted recombination, there
remains a need for specific, high-efficiency methods and
compositions for gene targeting. Besides making gene targeting more
readily available and practical, such improved methods and
compositions would also reduce side effects resulting from
non-targeted insertions. See, e.g., Hacien-Bey-Abina et al. (2003)
Science 302:415-419.
[0009] The RecA protein is the prototype of a family of prokaryotic
and eukaryotic proteins that catalyze genetic recombination (i.e.,
exchange of DNA sequence information between two DNA molecules).
RecA and its homologues participate in the repair of
double-stranded DNA breaks by catalyzing the synapsis of a
single-stranded DNA molecule with homologous sequences in a
double-stranded DNA to form a heteroduplex molecule. Branch
migration in the heteroduplex can result in the transfer of
sequence information from the single-stranded DNA to the
double-stranded molecule, as occurs in the processes of
recombination and gene conversion.
[0010] The remarkable and diverse activities of RecA have led
researchers to examine the use of this protein, and its homologues,
for stimulating homologous recombination and gene targeting in
eukaryotes. In tobacco, expression of bacterial RecA containing a
nuclear localization signal (NLS-RecA) increased resistance to
mytomycin C-induced DNA-crosslinking and also increased somatic
intrachromosomal recombination (recombination between homologous
chromosomes) by ten-fold. Reiss et al. (1996) Proc. Natl. Acad.
Sci. USA 93:3094-3098. In a separate study in tobacco, expression
of NLS-RecA was found to stimulate sister chromatid exchange
2.4-fold over wild-type levels. Reiss et al. (2000) Proc. Natl.
Acad. Sci. USA 97:3358-3363.
[0011] In mammalian cells, overexpression of NLS-RecA was reported
to stimulate gene targeting via homologous recombination 10-fold.
Shcherbakova et al. (2000) Mutation Res. 459:65-71. In human cells,
overexpression of the human RecA homologue RAD51 was able to
stimulate recombination by only 2 to 3-fold over wild type levels.
Yanez et al. (1999) Gene Ther. 6:1282-1290. Another study showed
that direct injection of preformed RecA-coated nucleoprotein
filaments into zebrafish embryos could correct a mutant form of the
enhanced green fluorescent protein (eGFP), albeit at a low
frequency. Cui et al. (2003) Marine Biotechnol. 5:174-184. In
similar injection experiments in zebrafish embryos, another group
showed that Rad52, a member of the Rad51 epistasis group, could
promote single-strand annealing and low level
oligonucleotide-mediated gene disruption. Takahashi et al. (2005)
Nucleic Acids Res. 33:e120. Other publications relating to RecA
include, e.g., U.S. Pat. No. 7,229,767. A recent disclosure
described the use of molecular tethers for targeted insertion of
transposon vectors, wherein the tether comprises a DNA-binding
domain that binds a target site in the vector, see U.S. Patent
Publication No. 2007/0031380. Other publications relating to
transposons include, for example, U.S. Pat. Nos. 6,498,458,
7,160,682, and 7,527,966.
[0012] Conventional tools used to perform reverse genetics and
create targeted modification of specific genes are limited to a few
species and require sophisticated and labor intensive technologies
that typically involve cloning or engineering of embryonic stem
cells. To address these limitations, innovative technologies were
developed that can be used to modify specific chromosomal regions
by direct injection of protein-nucleic acid complexes into
fertilized zygotes. A modified version of the bacterial RecA
protein is described that is able to promote homologous or
non-homologous recombination and insertion of exogenous DNA into
specific genomic locations for gene modification. This modified
version of RecA functions at frequencies several orders of
magnitude greater than previous reports. This is a highly active
form of RecA that functions in vertebrates and is expected to
function in animals and plants generally. The modification and use
of RecA is an unexpected and surprising result that was not
expected to function as it does, i.e., to promote homologous
recombination.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows the amino acid sequence of a NLS/RecA/Gal4
fusion protein (SEQ ID NO:3).
[0014] FIG. 2 shows the nucleotide sequence encoding a
NLS/RecA/Gal4 fusion protein (SEQ ID NO:4).
[0015] FIG. 3 shows the amino acid sequence of a NLS/RecA fusion
protein (SEQ ID NO:5).
[0016] FIG. 4 shows the nucleotide sequence encoding a NLS/RecA
fusion protein (SEQ ID NO:6).
[0017] FIG. 5 is an illustration of three types of RecA fusion
proteins.
[0018] FIG. 6 depicts results that show that injection of
complementary ssDNA-NLS-RecAGal4 filament leads to site-specific
insertion. As depicted, this filament causes loss of
herterozygosity (LOH) in heterozygous gol embryos, resulting in
mosaic eye pigmentation. Panel A: Genotype of the embryos used for
injection. All embryos were injected under the one-cell stage.
Panel B: Dorsal views of eyes at 3 dpf showing wild type
pigmentation patterns in a non-injected embryo (upper panel) and
mutant patterns (bottom panel) by injected gol
oligonucleotides-NLS-RecA-Gal4 targeting filaments. Injection of
targeting filament results in loss of heterozygosity at the gol
locus. Panel C: Four gol targeting probes coated by NLS-RecA-Gal4
are designed to target either exon4/5 or exon6 region. Gsg1 and
gsg2 probes carry stop codon mutation within the DNA probes which
show by filled circle. Gbg1 and gbg2 probes are 60 nt in length and
synthesized to adjacent genomic DNA sequences. These two probes do
not contain mutations.
[0019] FIG. 7 depicts results that show, for three different genes,
that gene expression is a result of site specific integration. As
depicted, expression of an EGFP reporter gene is consistent with
site specific integration into the gol, flh, and prominin-1 loci.
Single-stranded NLS-RecA-Gal4 filaments complementary to the (1)
gol, (2) flh, and (3) prominin-1 loci were co-injected with the
EGFP reporter gene cassette. EGFP expression consistent with
targeting gene expression was observed in 5-19% of the injected
embryos. For the gol gene, expression was observed in the eye, for
the flh gene in the notochord, and for the prominin-1 gene in the
dorsal diencephalon.
[0020] FIG. 8 depicts site-specific insertion of exogenous DNA. As
depicted, cssDNA-NLS-RecA-Gal4 filaments directed site-specific
insertion of GFP DNA. Panel A: Two regions of the gol gene were
amplified, denatured, and coated with NLS-RecA-Gal4 protein to make
ssDNA-NLS-RecA-Gal4 filaments. Filaments were injected with foreign
DNA containing a splice acceptor (SA) followed by the green
fluorescent protein (GFP) in three reading frames and a poly
adenylation signal (pA). Panel B: PCR amplification of junction
fragments between the foreign DNA and the endogenous gol locus were
obtained from DNA isolated from individual embryos. Bands marked
with a star were sequence-verified as junction fragments. Panel C:
Junction fragment map of insertions into the gol locus showing
insertion of exogenous DNA near ends of the regions complementary
to the junction fragments.
[0021] FIG. 9 depicts targeted mutation for creation of a
transgenic animal. The gol locus in the zebrafish germline was
targeted and modified. A testcross between a gol targeted founder
and gol.sup.b1 homozygotes produced offspring that fail to
complement the b1 allele (right) and its sibling with normal
pigment (left).
[0022] FIG. 10 depicts a model for gene targeting by
cssDNA-NLSRecAGal4. Both forward and reverse single strand (fss and
rss)-NLSRecAGal4 filaments are co-injected into zebrafish embryos.
The RecA homology search activity guides the filaments to the
targeted region. The cssDNA-NLSRecAGal4 filaments undergoes
homologous pairing and strand invasion, which causes the formation
of D-loops on the target chromosome. The structure is stabilized by
the Gal4 dimerization domains between the complementary filaments.
This compact DNA joint molecule is theorized to block replication
fork progression, leading to a double strand break (DSB).
DETAILED DESCRIPTION
[0023] Nucleic acids coated with certain fusion proteins were shown
to be targeted to specific target sites. Double stranded exogenous
nucleic acid sequences were incorporated into the host cell at the
target sites, and expressed by the cells. Animals thus transfected
continuously expressed the gene and passed the genetic alterations
to offspring. This method is very simple and powerful compared to
conventional technologies, which are limited to a few species and
require sophisticated and labor intensive techniques.
[0024] It was discovered that the pairing activity of RecA-DNA
filaments could be utilized to target biochemical activities to
specific chromosomal sites. Various filaments with chimeric RecA
proteins were tested with various genes. The vertebrate animal
model was the zebrafish. In this model, site-specific disruption of
a gene is demonstrated by inducing loss of heterozygosity (LOH) at
the golden locus in zebrafish after injection at the 1-cell stage.
LOH is visible by a mosaic pigmentation and was verified by direct
DNA analysis. The results reported herein demonstrate that DNA
filaments, of various sizes, coated with the fusion proteins are
able to cause site-directed mutations and targeted chromosomal
deletions in zebrafish that are transmitted to subsequent
generations. Further, co-injection of an exogenous nucleic acids
with the fusion protein promotes the insertion of the exogenous
nucleic acids into targeted genomic locations, likely through the
non-homologous end-joining pathway.
[0025] Without being limited to a particular theory, a model is
presented whereby the Gal4 domain of NLSRecAGal4 promotes the
dimerization of complementary single strand (css) filaments after
the filaments find their target. The model provides for a complex
that creates a steric block to replication, resulting in a stalled
replication fork and either repair of the locus or chromosomal
breakage.
[0026] Data herein provides evidence that proteinaceous fusion
molecules can be specifically targeted to a target site in a host
chromosome and create a break. This break can be exploited to
create mutants, discover gene function, insert exogenous genes, and
other purposes. Examples 1 and 2 details such a fusion molecule,
specifically, a NLS domain-RecA domain-Gal4 domain molecule or an
NLS domain-RecA domain, and DNA constructs for making them. These
fusion molecules retain nucleoprotein filament-forming function
(Example 3). In Example 4, the zebrafish model was used to
demonstrate disruption of specific gene sites. Double-stranded DNA
probes complementary to various sites in the gol locus were
denatured into single strands and formed into filaments with a
NLS/RecA/Gal4 proteinaceous fusion molecule. Both 1300 base pair
(bp) and 60 bp probes directed to distinct sites in gol were
successfully used. The probes were not integrated into the host
cell DNA.
[0027] Further data showed that exogenous DNA could then be
integrated at specific sites and that the method was generally
applicable to genes and not limited to gol. In Example 5, targeted
insertion events were demonstrated by the tissue-specific
expression of enhanced green fluorescent protein (EGFP) gene after
co-injection with ssDNA-NLS-RecA-Gal4 filaments complementary to
the gol, prominin1, and floating head (flh) loci (FIG. 7). The
expression was observed in the absence of an exogenous promoter,
i.e., the insertion site was chosen so as to take advantage of
native promoters and cellular machinery. Analysis of the insertion
sites showed them to be in the targeted gene, and within about 500
bp of the probe's target site. Finally, transfection of the
germline and progeny that expressed the exogenous genes was
demonstrated (Example 6). Biological mechanisms are detailed
below.
[0028] Other experiments were performed with a gol-mcherry-gol
replacement construct, which was able to target the gol genomic
locus. The gol gene exons are designated E1 through E9. The
targeting construct contained the mcherry (M) gene. Injection of
this replacement construct with NLSRecAGal4 resulted in red
fluorescent sectors in the eye. Furthermore, a junction fragment
was recovered after nested PCR amplification indicative of
homologous recombination between the gol replacement vector and the
endogenous gol gene.
[0029] Embodiments of the invention thus include a fusion molecule
with a recombinase domain and a DNA-binding domain. The fusion
molecule may include a nuclear localization signal or otherwise be
transported into the cell and nucleus. Systems may include probes
and exogenous DNA for insertion into a host cell. These features
are detailed herein.
[0030] Practice of the present disclosure employs, unless otherwise
indicated, standard methods and conventional techniques in the
fields of cell biology, developmental biology, reproductive
biology, molecular biology, biochemistry, cell culture, recombinant
DNA and related fields as are within the skill of the art. Such
techniques are described in the literature and thereby available to
those of skill in the art. See, for example, Alberts, B. et al.,
"Molecular Biology of the Cell," 5.sup.th edition, Garland Science,
New York, N.Y., 2008; Voet, D. et al. "Fundamentals of
Biochemistry: Life at the Molecular Level," 3.sup.rd edition, John
Wiley & Sons, Hoboken, N. J., 2008; Sambrook, J. et al.,
"Molecular Cloning: A Laboratory Manual," 3.sup.rd edition, Cold
Spring Harbor Laboratory Press, 2001; Ausubel, F. et al., "Current
Protocols in Molecular Biology," John Wiley & Sons, New York,
1987 and periodic updates; Freshney, R. I., "Culture of Animal
Cells: A Manual of Basic Technique," 4.sup.th edition, John Wiley
& Sons, Somerset, N J, 2000; and the series "Methods in
Enzymology," Academic Press, San Diego, Calif.
Fusion Molecules
[0031] The methods and compositions for targeted genome
modification disclosed herein involve, in certain embodiments, the
use of fusion molecules. For the purposes of the present
disclosure, a fusion molecule is a non-naturally-occurring molecule
that contains at least two domains joined to each other within a
single molecule, such that the two domains are not found together
in a naturally-occurring molecule. The domains can be
naturally-occurring or synthetic. The domains can be the same
chemical type of molecule, or can be different chemical types of
molecules. The term proteinaceous fusion molecule refers to a
fusion molecule having at least two polypeptide domains. Since it
has polypeptide domains it is "proteinaceous", and it may further
comprise non-protein features, e.g, polymeric linkers. The term
polypeptide domain refers to: a set of peptides joined together
that collectively and independently accomplish a biological
function. Examples of domains that satisfy this definition are zinc
fingers, the calcium-binding EF hand domain of calmodulin, peptide
sequences that exhibit specific binding to a predetermined target,
NLS, DNA-binding sequences, and portions of proteins that perform
the function of the wildtype protein (e.g., a derivative of RecA).
Polypeptide domains can thus, for example, be mixed-and-matched by
genetic engineering between one protein and another to make
chimeric proteins.
[0032] In certain embodiments, a proteinaceous fusion molecule
includes at least two domains selected from the group consisting of
a first domain that is a DNA-binding domain; e.g., a DNA-binding
protein or a functional fragment of a DNA-binding protein, a second
domain that comprises a polypeptide sequence having recombinase
activity, and a third domain that comprises a nuclear localization
signal. These domains may be in any order, e.g.,
NLS-recombinase-DNA binding or DNA-binding-NLS-recombinase. The
domains may be separated by linkers that are peptidic or made of
other materials.
[0033] In certain embodiments, each of the fusion molecule domains
corresponds to a distinct polypeptide sequence; for example a
polypeptide DNA-binding domain such as Gal4 and polypeptide
sequences from RecA having recombinase activity. However, it is
also possible for the fusion molecules to possess non-polypeptide
domains. For example, the DNA-binding domain can comprise a
polymer, a peptide spacer, a triplex-forming nucleic acid, a
polyamide, a minor groove binder, an intercalating agent, an
antibiotic and/or a nucleic acid.
[0034] Fusion molecules, including fusion proteins and nucleic
acids encoding them, are constructed by methods of cloning and
biochemical conjugation that are well-known to those of skill in
the art. Fusion proteins (and nucleic acids encoding them) may be
designed such that the translational reading frame is preserved
among the components of the fusion.
[0035] Fusions between polypeptide sequences possessing recombinase
activity, on the one hand, and a non-protein DNA-binding domain
(e.g., antibiotic, intercalator, minor groove binder, nucleic acid)
on the other, are constructed by methods of biochemical conjugation
known to those of skill in the art. See, for example, the Pierce
Chemical Company (Rockford, Ill.) Catalogue. In other embodiments,
a chemical linker is used to connect synthetically or recombinantly
produced domains. Such flexible linkers are known to persons of
skill in the art. For example, poly(ethylene glycol) linkers are
available from Shearwater Polymers, Inc. (Huntsville, Ala.). These
linkers optionally have amide linkages, sulfhydryl linkages, or
heterofunctional linkages.
[0036] Methods and compositions for making fusions between a minor
groove binder and a polypeptide have been described. Mapp et al.
(2000) Proc. Natl. Acad. Sci. USA 97:3930-3935. With respect to
fusion polypeptides, the term "operatively linked" refers to the
fact that each of the components performs the same function in
linkage to the other component as it would if it were not so
linked.
[0037] A functional fragment of a protein, polypeptide or nucleic
acid is a protein, polypeptide or nucleic acid whose sequence is
not identical to the full-length protein, polypeptide or nucleic
acid, yet retains the same function as the full-length protein,
polypeptide or nucleic acid. A functional fragment can possess
more, fewer, or the same number of residues as the corresponding
native molecule, and/or can contain one or more amino acid or
nucleotide substitutions. Methods for determining the function of a
nucleic acid (e.g., coding function, ability to hybridize to
another nucleic acid) are well-known in the art. Similarly, methods
for determining protein function are well-known. For example, the
DNA-binding function of a polypeptide can be determined, for
example, by filter-binding, electrophoretic mobility-shift, or
immunoprecipitation assays. See Ausubel et al., supra. The ability
of a protein to interact with another protein can be determined,
for example, by co-immunoprecipitation, two-hybrid assays or
complementation, either genetic or biochemical. See, for example,
Fields et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,585,245
and PCT WO 98/44350. Accordingly, embodiments include fusion
molecules with a functional fragment of one or more of a
recombinase, RecA, NLS, Gal4, and polypeptide DNA-binding
domains.
[0038] In certain embodiments, a fusion between a polypeptide
DNA-binding domain and polypeptide sequences possessing recombinase
activity is encoded by a fusion nucleic acid. In such cases, the
nucleic acid can be cloned into intermediate vectors for
transformation into prokaryotic or eukaryotic cells for replication
and/or expression. Intermediate vectors for storage or manipulation
of the fusion nucleic acid or production of fusion protein can be
prokaryotic vectors, (e.g., plasmids), shuttle vectors, insect
vectors, or viral vectors for example. A fusion nucleic acid can
also cloned into an expression vector, for administration to a
bacterial cell, fungal cell, protozoal cell, plant cell, or animal
cell, e.g., a mammalian cell or a human cell. Vectors for
replication, expression, storage and/or manipulation of cloned
nucleic acid sequences are well-known in the art. See, e.g.,
Sambrook, supra, and Ausubel, supra.
[0039] Thus, expression of a fusion protein in a cell can result
from delivery of the fusion protein to the cell or by delivery of a
polynucleotide encoding the fusion protein to a cell, wherein the
polynucleotide is transcribed, and the transcript is translated, to
generate the fusion protein. Trans-splicing, polypeptide cleavage
and polypeptide ligation can also be involved in expression of a
protein in a cell. Methods for polynucleotide and polypeptide
delivery to cells are presented elsewhere in this disclosure.
[0040] Linker domains can be included between polypeptide domains,
e.g., between a DNA-binding domain and polypeptide sequences having
recombinase activity. Such linkers can be polypeptide sequences,
such as poly-glycine sequences of from 1 to about 200 amino acids.
Linker domains can comprise flexible amino acid subsequences which
are synthesized as part of a recombinant fusion protein. For
example, a linker domain can comprise amino acid sequence with a
plurality of amino acids, e.g, from 1 to 20; artisans will
immediately appreciate that all the ranges and values within the
explicitly stated ranges are contemplated, e.g., 2, or 10, or from
3 to 11. Alternatively, flexible linkers can be rationally designed
using computer programs capable of modeling both DNA-binding sites
and the peptides themselves (Desjarlais & Berg (1993) Proc.
Natl. Acad. Sci. USA 90:2256-2260; Desjarlais et al. (1994) Proc.
Natl. Acad. Sci. USA 91:11099-11103) or by phage display methods.
Methods for obtaining sequences that mediate non-covalent linkage
between polypeptide domains have also been described. Wang et al.
(1999) Proc. Natl. Acad. Sci. USA 96:9568-9573.
Nuclear Localization Signals
[0041] Fusion molecules, as disclosed herein, also optionally
comprise nuclear localization signals ("NLS"). As used herein, the
term "nuclear localization signal" means an amino acid sequence
known to, in vivo, direct a protein disposed in the cytoplasm of a
cell across the nuclear membrane and into the nucleus of the cell.
A nuclear localization signal can also target the exterior surface
of a cell. Thus, a single nuclear localization signal can direct
the entity with which it is associated to the exterior of a cell
and to the nucleus of a cell. Such sequences can be of any size and
composition, for example between 4 and 400 amino acids; artisans
will immediately appreciate that all the ranges and values within
the explicitly stated ranges are contemplated, for example more
than 25, 25, 15, 12, 10, 8, 7, 6, 5 or 4 amino acids.
[0042] NLS are peptidic groups that signal importation of a protein
into the nucleus. Examples of NLS are SV40 large T-antigen,
nucleoplasmin, HIV-1 Rev, and hnRNPA1 (M9), see Escriou et al., NLS
bioconjugates for targeting therapeutic genes to the nucleus,
Advanced Drug Delivery Reviews, 55 (2003) 295-306. Several peptides
have been derived from the SV40 T antigen. These include a short
NLS or long NLS's. Other NLS peptides have been derived from M9
protein, nucleoplasmin, and c-myc.
DNA-Binding Domains
[0043] In certain embodiments, the compositions and methods
disclosed herein involve fusions between a DNA-binding domain and a
domain having recombinase activity. Any DNA-binding domain known in
the art can be used as part of a fusion molecule. A DNA-binding
domain can comprise any molecular entity capable of
sequence-specific binding to chromosomal DNA. Binding can be
mediated by electrostatic interactions, hydrophobic interactions,
or any other type of chemical interaction. Examples of moieties
which can comprise part of a DNA-binding domain include, but are
not limited to, minor groove binders, major groove binders,
antibiotics, intercalating agents, peptides, polypeptides, peptide
nucleic acids, polyamides, oligonucleotides, and polynucleotides.
An example of a DNA-binding nucleic acid is a triplex-forming
oligonucleotide.
[0044] Embodiments include fusion molecules with a DNA-binding
domain that are directed to techniques and treatments for gene
conversion, homology-independent gene targeting, homologous
recombination, targeted mutagenesis, genetic diseases, transgenic
animals, expression vectors, and administration into plants.
[0045] Minor groove binders include substances which, by virtue of
their steric and/or electrostatic properties, interact
preferentially with the minor groove of double-stranded nucleic
acids. Certain minor groove binders exhibit a preference for
particular sequence compositions. For instance, netropsin,
distamycin and CC-1065 are examples of minor groove binders which
bind specifically to AT-rich sequences, particularly runs of A or
T. WO 96/32496.
[0046] Polyamide DNA-binding domains are described, for example, in
U.S. Pat. No. 6,555,692. Peptide nucleic acids are described, for
example, in U.S. Pat. Nos. 5,539,082; 5,773,571; 6,395,474;
6,451,968 and 7,378,485. See also Nielsen et al. (1991) Science
254:1497-1500.
[0047] Many antibiotics are known to exert their effects by binding
to DNA. Binding of antibiotics to DNA is often sequence-specific or
exhibits sequence preferences. Actinomycin, for instance, is a
relatively GC-specific DNA binding agent.
[0048] Polypeptide DNA binding domains are found, for example, in
proteins involved in DNA replication, DNA repair, recombination and
transcription. Defined regions within the polypeptide sequence of
various transcription factors have been shown to be responsible for
sequence-specific binding to DNA. These regions include, but are
not limited to, motifs known as leucine zippers, helix-loop-helix
(HLH) domains, helix-turn-helix domains, zinc fingers, beta-sheet
motifs, steroid receptor motifs, bZIP domains homeodomains,
AT-hooks and others. The amino acid sequences of these motifs are
known and, in some cases, amino acids that are critical for
sequence specificity have been identified. See, for example, Pabo
et al. (1992) Ann. Rev. Biochem. 61:1053-1095 and references cited
therein. Exemplary well-characterized DNA-binding domains include
those for LexA, Gal4 and zif268. Webster et al., (1988) Cell 52:
169-178.
[0049] Peptide sequences involved in specific DNA recognition, such
as those found in transcription factors, can be obtained through
recombinant DNA cloning and expression techniques or by chemical
synthesis, and can be attached to other components of a fusion
molecule by methods known in the art.
[0050] In addition to naturally-occurring DNA-binding domains such
as those described above, non-naturally-occurring, engineered
DNA-binding domain can also be used. In this regard, the zinc
finger DNA-binding domain is useful, inasmuch as it is possible to
engineer zinc finger proteins to bind to any DNA sequence of
choice. A zinc finger binding domain comprises one or more zinc
finger structures. Miller et al. (1985) EMBO J 4:1609-1614; Rhodes
(1993) Scientific American February: 56-65; U.S. Pat. No.
6,453,242. Typically, a single zinc finger is about 30 amino acids
in length and contains four zinc-coordinating amino acid residues.
Structural studies have demonstrated that the canonical
(C.sub.2H.sub.2) zinc finger motif contains two beta sheets (held
in a beta turn which generally contains two zinc-coordinating
cysteine residues) packed against an alpha helix (generally
containing two zinc coordinating histidine residues).
[0051] Zinc fingers include both canonical C.sub.2H.sub.2 zinc
fingers (i.e., those in which the zinc ion is coordinated by two
cysteine and two histidine residues) and non-canonical zinc fingers
such as, for example, C.sub.3H zinc fingers (those in which the
zinc ion is coordinated by three cysteine residues and one
histidine residue) and C.sub.4 zinc fingers (those in which the
zinc ion is coordinated by four cysteine residues). Non-canonical
zinc fingers can also include those in which an amino acid other
than cysteine or histidine is substituted for one of these
zinc-coordinating residues. See e.g., WO 02/057293 (Jul. 25, 2002)
and US 2003/0108880 (Jun. 12, 2003).
[0052] Zinc finger binding domains can be engineered to bind to a
sequence of choice. See, for example, Beerli et al. (2002) Nature
Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.
70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660;
Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al.
(2000) Curr. Opin. Struct. Biol. 10:411-416. Consequently, zinc
finger binding domain can be engineered to have a novel binding
specificity, compared to a naturally-occurring zinc finger protein.
Engineering methods include, but are not limited to, rational
design and various types of empirical selection methods. Rational
design includes, for example, using databases comprising triplet
(or quadruplet) nucleotide sequences and individual zinc finger
amino acid sequences, in which each triplet or quadruplet
nucleotide sequence is associated with one or more amino acid
sequences of zinc fingers which bind the particular triplet or
quadruplet sequence. See, for example, U.S. Pat. Nos. 6,140,081;
6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617;
U.S. Patent Application Publication Nos. 2002/0165356;
2004/0197892; 2007/0154989; 2007/0213269; and International Patent
Application Publication Nos. WO 98/53059 and WO 2003/016496.
[0053] Exemplary selection methods, including phage display,
interaction trap, hybrid selection and two-hybrid systems, are
disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988;
6,013,453; 6,140,466; 6,200,759; 6,242,568; 6,410,248; 6,733,970;
6,790,941; 7,029,847 and 7,297,491; as well as U.S. Patent
Application Publication Nos. 2007/0009948 and 2007/0009962; WO
98/37186; WO 01/60970 and GB 2,338,237.
[0054] Additional methods for design of sequence-specific zinc
finger DNA-binding domains have been described by Maeder et al.
(2008) Mol. Cell 31:294-301.
[0055] Enhancement of binding specificity for zinc finger binding
domains has been described, for example, in U.S. Pat. No. 6,794,136
(Sep. 21, 2004). Additional aspects of zinc finger engineering,
with respect to inter-finger linker sequences, are disclosed in
U.S. Pat. No. 6,479,626 and U.S. Patent Application Publication No.
2003/0119023. See also Moore et al. (2001a) Proc. Natl. Acad. Sci.
USA 98:1432-1436; Moore et al. (2001b) Proc. Natl. Acad. Sci. USA
98:1437-1441 and WO 01/53480.
[0056] Zinc finger DNA-binding domains, engineered to bind a DNA
sequence of choice, are commercially available (CompoZr.TM.,
Sigma-Aldrich, St. Louis, Mo.). Fusions between a recombinase
domain and a zinc finger DNA-binding domain have been described by
Akopian et al. (2003) Proc. Natl. Acad. Sci. USA 100:8688-8691.
[0057] All of the references cited in this section, entitled "DNA
Binding Domains," are hereby incorporated by reference herein in
their entireties for the purposes of disclosing art-recognized
DNA-binding domains and methods for the design, selection and
engineering of zinc finger DNA-binding domains.
[0058] In general, Gal4 is used as an example of a DNA-binding
domain. Similarly, RecA is used as an example of a recombinase,
with a fusion protein of the two being an exemplary fusion protein.
One of the utilities of the NLS-RecA-Gal4 fusion protein is its
ability like RecA to coat single-stranded DNA and find homologous
regions in a genome to the RecA filament. By doing this, the RecA
part of NLS-RecA-Gal4 brings the activity associated with the Gal4
DNA binding domain into the targeted chromosomal region. The Gal4
DNA binding motif contains both a metal coordination center and a
dimerization motif. Both of these activities are like required for
NLS-RecA-Gal4 fusion protein coated DNA to promote chromosomal
breaks by potentially creating stalled replication forks. Other
mechanisms are envisioned as well.
[0059] Using this ability of RecA fusion proteins, different
activities can be carried to distinct chromosomal locations by
substituting the Gal4 domain with other proteins or motifs. For
example, a nuclease could be substituted for Gal4. In this case the
RecA-coated filament would bring the nuclease, via its attachment
to RecA, to a specific chromosomal site. Several recent reports
have highlighted the use of zinc finger nucleases to modify
specific chromosomal regions by their ability to induce double
strand breaks (Bibikova et al., 2002; Porteus and Baltimore, 2003;
Urnov et al., 2005; Wright et al., 2005). The specificity of this
technique relies on the observation that the restriction
endonuclease, FokI, is only active as a dimer. Consequently, this
system requires that the FokI-zinc fingers bind two distinct sites.
The FokI-induced double strand breaks can be repaired from an
exogenously supplied plasmid DNA that contains a region of
homology. If the exogenously supplied DNA contains a change
relative to the chromosomal target, this change can be incorporated
into the repaired chromosome. This technique appears to function
well in a variety of systems and has been used efficiently to
modify chromosomes in Drosophila (Bibikova et al., 2002), tobacco
(Wright et al., 2005), and human cells (Porteus and Baltimore,
2003; Urnov et al., 2005). Because the engineering of zinc fingers
can require significant selection, the widespread use of this
technique may be limited. The homology searching function of RecA
can substitute for the zinc fingers in this system. Either chimeric
RecA-FokI proteins or RecA coated filaments that bind Fok1 could
induce specific double strand breaks at specific chromosomal sites.
Other nucleases such as I-sce-I and EcoRI could also be used.
[0060] Other activities that could replace the Gal4 domain include
different DNA binding motifs such as zinc fingers and
helix-turn-helix proteins. This would further promote distinct
activities and the ability to site specifically modify a
chromosome.
[0061] Techniques include introduction of a fusion protein with a
DNA-binding domain that is not specifically bound to a DNA. Trivial
binding events are not specific binding events. Specific binding,
as that term is commonly used in the biological arts, generally
refers to a molecule that binds to a target with a relatively high
affinity compared to non-target tissues, and generally involves a
plurality of non-covalent interactions, such as electrostatic
interactions, van der Waals interactions, hydrogen bonding, and the
like. Specific binding interactions characterize antibody-antigen
binding, enzyme-substrate binding, and specifically binding
protein-receptor interactions; while such molecules may bind
tissues besides their targets from time to time, such binding is
said to lack specificity and is not specific binding. Thus a
DNA-binding domain will not exhibit specific binding to a nucleic
acid unless a sequence specifically recognized by the domain is
present.
Recombinases
[0062] The term recombinase refers to a genetic recombination
enzyme that enzymatically catalyzes, in a cell, the joining of
relatively short pieces of DNA between two relatively longer DNA
strands. Recombinases include Cre recombinase, Hin recombinase,
RecA, RAD51, Tre, and FLP. Cre recombinase is a Type I
topoisomerase from P1 bacteriophage that catalyzes site-specific
recombination of DNA between loxP sites. Hin recombinase is a 21 kD
protein composed of 198 amino acids that is found in the bacteria
Salmonella. Hin belongs to the serine recombinase family of DNA
invertases in which it relies on the active site serine to initiate
DNA cleavage and recombination. RAD51 is a human gene. The protein
encoded by this gene is a member of the RAD51 protein family which
assist in repair of DNA double strand breaks. RAD51 family members
are homologous to the bacterial RecA and yeast Rad51. Tre
recombinase is an experimental enzyme that in lab tests has
successfully removed DNA inserted by HIV from infected cells. The
enzyme was derived from Cre recombinase through selective mutation
for the purposes of identifying HIV markers, which are not bounded
by loxP sites and therefore disallow attempts at Cre-Lox
recombination. FLP refers to Flippase recombination enzyme (FLP or
Flp) derived from the 2.mu. plasmid of the baker's yeast
Saccharomyces cerevisiae.
[0063] RecA is known for its recombinase activity to catalyze
strand exchange during the repair of double-strand breaks by
homologous recombination (McGrew and Knight, 2003) Radding, et al.,
1981; Seitz et al., 1998). RecA has also been shown to catalyze
proteolysis, e.g., of the LexA and .lamda. repressor proteins, and
to possess DNA-dependent ATPase activity. After a double-strand
break occurs from ionizing radiation or some other insult,
exonucleases chew back the DNA ends 5' to 3', thereby exposing one
strand of the DNA (Cox, 1999; McGrew and Knight, 2003). The
single-stranded DNA becomes stabilized by single-strand binding
protein (SSB). After binding of SSB, RecA binds the single-stranded
(ss) DNA and forms a helical nucleoprotein filament (referred to as
a filament or a presynaptic filament). During DNA repair, the
homology-searching functions of RecA direct the filament to
homologous DNA and catalyze homologous base pairing and strand
exchange. This results in the formation of DNA heteroduplex. After
strand invasion, DNA polymerase elongates the ssDNA based on the
homologous DNA template to repair the DNA break, and crossover
structures or Holliday junctions are formed. RecA also shows a
motor function that participates in the migration of the crossover
structures (Campbell and Davis, 1999).
[0064] Recombinase activity comprises a number of different
functions. For example, polypeptide sequences having recombinase
activity are able to bind in a non-sequence-specific fashion to
single-stranded DNA to form a nucleoprotein filament. Such
recombinase-bound nucleoprotein filaments are able to interact in a
non-sequence-specific manner with a double-stranded DNA molecule,
search for sequences in the double-stranded molecule that are
homologous to sequences in the filament, and, when such sequences
are found, displace one of the strands of the double-stranded
molecule to allow base-pairing between sequences in the filament
and complementary sequences in one of the strands of the double
stranded molecule. Such steps are collectively denoted
"synapsis."
[0065] RecA and RecA-like proteins (called Rad51 in non-bacterial
species) have been examined for stimulating gene targeting and
homologous recombination in a variety of eukaryotic systems. In
tobacco cells, expression of bacterial RecA containing a nuclear
localization signal (NLS) increases the repair of mitomycin
C-induced DNA damage by homologous recombination and somatic
intrachromosomal recombination (recombination between homologous
chromosomes) from three to ten fold (Reiss et al., 1996).
Expression of NLSRecA in tobacco can also stimulate sister
chromatid exchange 2.4-fold over wild-type levels (Reiss et al.,
2000). In somatic mammalian cells, overexpression of NLSRecA
stimulates gene targeting by homologous recombination 10-fold
(Shcherbakova et al., 2000). However, in human cells,
overexpression of a human homologue of RecA, hRAD51, only
stimulates recombination 2 to 3-fold over wild type levels under
the antibiotic selection (Yanez and Porter, 1999). In zebrafish, a
mutant form of the enhanced green fluorescent protein (EGFP) was
corrected at low frequency by injecting ssDNA-RecA filaments
directly (Cui et al., 2003). Rad52, a member of the Rad51 epistasis
group, also promotes single-strand annealing and low level gene
disruption in zebrafish using mutated oligonucleotides (Takahashi
and Dawid, 2005). Taken together, these studies indicate that
ectopic expression of RecA or Rad51 results in a modest stimulation
of homologous recombination but does not increase levels enough to
be useful for gene targeting.
[0066] Thus recombinase activities include, but are not limited to,
single-stranded DNA-binding, synapsis, homology searching, duplex
invasion by single-stranded DNA, heteroduplex formation, ATP
hydrolysis and proteolysis. The prototypical recombinase is the
RecA protein from E. coli. See, for example, U.S. Pat. No.
4,888,274. Prokaryotic RecA-like proteins have also been described
in Salmonella, Bacillus and Proteus species. A thermostable RecA
protein, from Therms aquaticus, has been described in U.S. Pat. No.
5,510,473. A bacteriophage T4 homologue of RecA, the UvsX protein,
has been described. RecA mutants, having altered recombinase
activities, have been described, for example, in U.S. Pat. Nos.
6,774,213; 7,176,007 and 7,294,494. Plant RecA homologues are
described in, for example, U.S. Pat. Nos. 5,674,992; 6,388,169 and
6,809,183. RecA fragments containing recombinase activity have been
described, for example, in U.S. Pat. No. 5,731,411. Mutant RecA
proteins having enhanced recombinase activity such as, for example,
RecA803 have been described. See, for example, Madiraju et al.
(1988) Proc. Natl. Acad. Sci. USA 85:6592-6596.
[0067] A eukaryotic homologue of RecA, also possessing recombinase
activity, is the Rad51 protein, first identified in the yeast
Saccharomyces cerevisiae. See Bishop et al., (1992) Cell 69: 439-56
and Shinohara et al, (1992) Cell: 457-70 Aboussekhra, et al.,
(1992) Mol. Cell. Biol. 72, 3224-3234. Basile et al., (1992) Mol.
Cell. Biol. 12, 3235-3246. Plant Rad51 sequences are described in
U.S. Pat. Nos. 6,541,684; 6,720,478; 6,905,857 and 7,034,117.
Another yeast protein that is homologous to RecA is the Dmc1
protein. RecA/Rad51 homologues in organisms other than E. coli and
S. cerevisiae have been described. Morita et al. (1993) Proc. Natl.
Acad. Sci. USA 90:6577-6580; Shinohara et al. (1993) Nature Genet.
4:239-243; Heyer (1994) Experientia 50:223-233; Maeshima et al.
(1995) Gene 160:195-200; U.S. Pat. Nos. 6,541,684 and
6,905,857.
[0068] Herein, "RecA" or "RecA protein" refers to a family of
RecA-like recombination proteins having essentially all or most of
the same functions, particularly: (i) the ability to position
properly oligonucleotides or polynucleotides on their homologous
targets for subsequent extension by DNA polymerases; (ii) the
ability topologically to prepare duplex nucleic acid for DNA
synthesis; and, (iii) the ability of RecA/oligonucleotide or
RecA/polynucleotide complexes efficiently to find and bind to
complementary sequences. The best characterized RecA protein is
from E. coli; in addition to the original allelic form of the
protein a number of mutant RecA-like proteins have been identified,
for example, RecA803. Further, many organisms have RecA-like
strand-transfer proteins including, for example, yeast, drosophila,
mammals including humans, and plants. These proteins include, for
example, Rec1, Rec2, Rad51, Rad51B, Rad51C, Rad51D, Rad51E, XRCC2
and DMC1. An embodiment of the recombination protein is the RecA
protein of E. coli. Alternatively, the RecA protein can be the
mutant RecA-803 protein of E. coli, a RecA protein from another
bacterial source or a homologous recombination protein from another
organism.
[0069] Additional descriptions of proteins having recombinase
activity are found, for example, in Fugisawa et al. (1985) Nucl.
Acids Res. 13:7473; Hsieh et al. (1986) Cell 44:885; Hsieh et al.
(1989) J. Biol. Chem. 264:5089; Fishel et al. (1988) Proc. Natl.
Acad. Sci. USA 85:3683; Cassuto et al. (1987) Mol. Gen. Genet.
208:10; Ganea et al. (1987) Mol. Cell Biol. 7:3124; Moore et al.
(1990) J. Biol. Chem.:11108; Keene et al. (1984) Nucl. Acids Res.
12:3057; Kimiec (1984) Cold Spring Harbor Symp. 48:675; Kimeic
(1986) Cell 44:545; Kolodner et al. (1987) Proc. Natl. Acad. Sci.
USA 84:5560; Sugino et al. (1985) Proc. Natl. Acad, Sci. USA 85:
3683; Halbrook et al. (1989) J. Biol. Chem. 264:21403; Eisen et al.
(1988) Proc. Natl. Acad. Sci. USA 85:7481; McCarthy et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5854; and Lowenhaupt et al. (1989) J.
Biol. Chem. 264:20568, which are incorporated herein by reference.
See also Brendel et al. (1997) J. Mol. Evol. 44:528 541.
[0070] Examples of proteins having recombinase activity include
recA, recA803, uvsX, and other recA mutants and recA-like
recombinases (Roca (1990) Crit. Rev. Biochem. Molec. Biol. 25:415),
sep1 (Kolodner et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:5560;
Tishkoff et al. (1991) Molec. Cell. Biol. 11:2593), RuvC
(Dunderdale et al. (1991) Nature 354:506), DST2, KEM1 and XRN1
(Dykstra et al. (1991) Molec. Cell. Biol. 11:2583), STPa/DST1
(Clark et al. (1991) Molec. Cell. Biol. 11:2576), HPP-1 (Moore et
al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:9067), other eukaryotic
recombinases (Bishop et al. (1992) Cell 69:439; and Shinohara et
al. (1992) Cell 69:457); incorporated herein by reference.
[0071] In vitro-evolved proteins having recombinase activity have
been described in U.S. Pat. No. 6,686,515. Further publications
relating to recombinases include, for example, U.S. Pat. Nos.
7,732,585, 7,361,641, 7,144,734. For a review of recombinases, see
Cox (2001) Proc. Natl. Acad. Sci. USA 98:8173-8180.
Methods for Forming Nucleoprotein Filaments
[0072] In certain embodiments, a fusion molecule as disclosed
herein is contacted with a nucleic acid to form a nucleoprotein
filament, or "filament". The term filament, in the context of
forming a structure with a recombinase, is a term known to artisans
in these fields. The nucleoprotein filament so formed can then be,
e.g., contacted with another nucleic acid or introduced into a
cell. Methods for forming nucleoprotein filaments, wherein the
filaments comprise polypeptide sequences having recombinase
activity and a nucleic acid, are well-known in the art. See, e.g.,
Cui et al. (2003) Marine Biotechnol. 5:174-184 and U.S. Pat. Nos.
4,888,274; 5,763,240; 5,948,653 and 7,199,281, the disclosures of
which are incorporated by reference for the purposes of disclosing
exemplary techniques for binding recombinases to nucleic acids to
form nucleoprotein filaments.
[0073] In general, a molecule having recombinase activity is
contacted with a linear, single-stranded nucleic acid. The linear,
single-stranded nucleic acid may be a probe. The preparation of
such single stranded nucleic acids are known. The reaction mixture
typically contains a magnesium ion. Optionally, the reaction
mixture is buffered and optionally also contains ATP, dATP or a
nonhydrolyzable ATP analogue, such as, for example,
.gamma.-thio-ATP (ATP-.gamma.-S) or .gamma.-thio-GTP
(GTP-.gamma.-S). Reaction mixtures can also optionally contain an
ATP-generating system. Double-stranded DNA molecules can be
denatured (e.g., by heat or alkali) either prior to, or during,
filament formation. Optimization of the molar ratio of recombinase
to nucleic acid is within the skill of the art. For example, a
series of different concentrations of recombinase can be added to a
constant amount of nucleic acid, and filament formation assayed by
mobility in an agarose or acrylamide gel. Because bound protein
retards the electrophoretic mobility of a polynucleotide, filament
formation is evidenced by retarded mobility of the nucleic acid.
Either maximum degree of retardation, or maximum amount of nucleic
acid migrating with a retarded mobility, can be used to indicate
optimal recombinase:nucleic acid ratios. Protein-DNA association
can also be quantitated by measuring the ability of a
polynucleotide to bind to nitrocellulose.
Exogenous Sequences
[0074] The methods and compositions set forth herein can be used
for targeted integration of exogenous sequences (also referred to
herein as donor sequences) into a region of interest in the genome
of a cell. Targeted integration of an exogenous sequence can occur
by both homology-dependent and homology-independent mechanisms. The
data provided herein show that broad applicability for these
techniques across species and for broad incorporation of DNAs
generally. Accordingly, embodiments include insertion of DNAs to
treat the various conditions described herein, as well as therapies
to insert wild-type non-defective DNAs into cells to replace
defective nucleic acid sequences. Thus embodiments include
exogenous nucleic acids directed to techniques and treatments for
gene conversion, homology-independent gene targeting, homologous
recombination, targeted mutagenesis, genetic diseases, transgenic
animals, expression vectors, and administration into plants.
[0075] Exemplary exogenous sequences include, but are not limited
to, cDNAs, promoter sequences, enhancer sequences, epitope tags,
marker genes, cleavage enzyme recognition sites and various types
of expression constructs. Marker genes include, but are not limited
to, sequences encoding proteins that mediate antibiotic resistance
(e.g., ampicillin resistance, neomycin resistance, G418 resistance,
puromycin resistance), sequences encoding colored or fluorescent or
luminescent proteins (e.g., green fluorescent protein, enhanced
green fluorescent protein, red fluorescent protein, luciferase),
and proteins which mediate enhanced cell growth and/or gene
amplification (e.g., dihydrofolate reductase). Epitope tags
include, for example, one or more copies of FLAG, His, myc, Tap, HA
or any detectable amino acid sequence.
[0076] Protein expression constructs optionally include, e.g.,
cDNAs and transcriptional control sequences in operative linkage
with cDNA sequences. Transcriptional control sequences include
promoters, enhancers and insulators. Additional transcriptional and
translational regulatory sequences which can be included in
expression constructs include, e.g., internal ribosome entry sites,
sequences encoding 2A peptides and polyadenylation signals. For
optimal expression of one or more proteins encoded by exogenous
sequences integrated into a genome, the chromosomal integration
site should be compatible with high-level transcription of the
integrated sequences, preferably in a wide range of cell types and
developmental states. However, it has been observed that
transcription of integrated sequences varies depending on the
integration site due to, among other things, the chromatin
structure of the genome at the integration site. Accordingly,
genomic target sites that support high-level transcription of
integrated sequences are desirable. Non-limiting examples of
chromosomal regions that do not encode an essential gene and
support high-level transcription of sequences integrated therein
include the murine Rosa26 locus (and its human homologue), the
human CCR5 locus and the AAV P1 integration site on human
chromosome 19. Additional genomic target sites supporting
high-level transcription of integrated sequences can be identified
as regions of open chromatin as described, for example in U.S.
Patent Application Publications 2002/0064802 (May 30, 2002) and
2002/0081603 (Jun. 27, 2002).
[0077] Cleavage enzyme recognition sites include, for example,
sequences recognized by restriction endonucleases, homing
endonucleases and/or meganucleases. Targeted integration of a
cleavage enzyme recognition site (by either homology-dependent or
homology-independent mechanisms) is useful for generating cells
whose genome contains only a single site that can be cleaved by a
particular enzyme. Contacting such cells with an enzyme that
recognizes and cleaves at the single site facilitates subsequent
targeted integration of exogenous sequences (by either
homology-dependent or homology-independent mechanisms) and/or
targeted mutagenesis at the site that is cleaved.
[0078] For certain embodiments, it is desirable that an integration
site is not present in an essential gene (e.g., a gene essential
for cell viability), so that inactivation of said essential gene
does not result from integration of the exogenous sequences. On the
other hand, if the intent is to disable gene function (i.e., create
a gene "knock-out") targeted integration of an exogenous sequence
to disrupt an endogenous gene is an effective method. In these
cases, the exogenous sequence can be any sequence capable of
blocking transcription of the endogenous gene or of generating a
non-functional translation product, for example a short patch of
amino acid sequence, which is optionally detectable (see above). In
certain embodiments, the exogenous sequences can comprise a marker
gene (described above), allowing selection of cells that have
undergone targeted integration. In certain embodiments, it will
also be desirable that integration of exogenous sequences not
result in ectopic activation of one or more cellular genes (e.g.,
oncogenes). On the other hand, in the case of integration of
promoter and/or enhancer sequences, ectopic expression may be
desired.
[0079] In certain embodiments, targeted integration is used to
insert a RNA expression construct, e.g., sequences responsible for
regulated expression of micro RNA, siRNA or shRNA. Promoters,
enhancers and additional transcription regulatory sequences, as
described above, can also be incorporated in a RNA expression
construct
Probes
[0080] The data presented herein shows that a probe may be
associated with a nucleoprotein filament to direct the filament
with specificity to a target on a host cell chromosome. A target
refers to a predetermined molecule, tissue, or location that the
user intends to bind with the probe. A probe, in the context of a
nucleoprotein filament, refers to a nucleic acid with
complementarity to a target nucleic acid sequence. Artisans are
familiar with methods for identifying sites of interest and
developing probes. Probes may be chosen as suited to the
recombinase chosen. The size of the probe may, accordingly be
chosen. Examples include probes with 60 bp or 1300 bp, or with a
length in the range of about 10 and about 10,000 residues; artisans
will immediately appreciate that all the ranges and values within
the explicitly stated ranges are contemplated, e.g., from about 40
to about 5,000, from about 60 to about 1300, from about 10 to about
3000, at least 10, at least about 30.
[0081] The probes may be chosen with the degree of specificity
intended. As demonstrated herein, exogenous sequences may be placed
with a high degree of reproducible accuracy. The sequences may be
placed in the targeted gene. The specificity of placement may be
measured in basepairs (bp) by comparing the most upstream point of
the probe to the most upstream point of the inserted exogenous
sequence, with the difference between these two points being the
distance from the probe to the site of insertion. Accordingly,
exogenous nucleic acids may be placed, and probes may be designed
for placement, with a predetermined specificity; for example,
between about 200 to about 2000 bp. Artisans will immediately
appreciate that all the ranges and values within the explicitly
stated ranges are contemplated, e.g., less than about 500 bp, less
than about 1000 bp, less than about 5000 bp, or from about 500 to
less than about 5000 bp. A predetermined specificity may be
measured in vitro using the zebrafish animal model and following
the procedures in the Examples, with directly injected zebrafish
embryos incorporating an exogenous DNA within the stated range with
90% accuracy as measured for the embryos that are successfully
transfected.
[0082] Embodiments include probes directed to techniques and
treatments for gene conversion, homology-independent gene
targeting, homologous recombination, targeted mutagenesis, genetic
diseases, transgenic animals, expression vectors, and
administration into plants.
Applications
[0083] Because recombinases are strongly conserved, among both
eukaryotes and prokaryotes, and because the recombination-promoting
activity of the fusion proteins disclosed herein does not depend
upon the presence of a binding site for the sequence-specific
DNA-binding domain present in the fusion protein, the disclosed
methods and compositions will be widely applicable in many species.
These include, but are not limited to, prokaryotes, eukaryotes,
plants, metazoans, vertebrates, mammals and humans. Plants include
monocotyledonous and dicotyledonous species. Exemplary plants
include Arabadopsis. Exemplary metazoans include fruit flies
(Drosophila), roundworms (Caenorhabditis). Exemplary vertebrates
include frogs (e.g., Xenopus) fish (e.g., Danio). Exemplary mammals
include bovines, porcines, ovines, caprines, equines, felines,
canines, murines, and humans.
[0084] The methods and compositions disclosed herein will find use
in both research and therapeutic applications, as will now be
described.
Gene Conversion
[0085] In certain embodiments, introduction, into a cell, of a
fusion molecule comprising a recombinase domain and a
sequence-specific DNA-binding domain leads to an overall,
genome-wide, increase in recombinational events, which can be
manifested as gene conversion or loss of heterozygosity. Selection
of a recombinational event of interest allows the isolation of
novel sequences, including, for example, different alleles or
haplotypes of genomic sequences, mutant sequences, wild-type
sequences, insertions, deletions or rearrangements.
[0086] The DNA cleavage activity of a recombinase domain can be
targeted by formation of a nucleoprotein filament containing a
fusion molecule comprising the recombinase domain, as disclosed
herein, and a sequence homologous to a genomic sequence of
interest. Such fusion molecules, when introduced into a cell, can
facilitate targeted mutagenesis in a genomic region of interest
resulting from cleavage in the region of interest followed by
non-homologous end-joining. Such mutations can result, for example,
in gene knock-outs, e.g., for functional genomics or target
validation.
[0087] Targeted DNA cleavage, mediated either by a fusion molecule
as disclosed herein or by a nucleoprotein filament as disclosed
herein, conducted in the absence of an exogenous polynucleotide
(preferably in S or G.sub.2 phase), can also stimulate
recombination between homologous chromosomes.
Homologs-Independent Gene Targeting
[0088] Integration of exogenous sequences at a region of interest
in a genome, when the exogenous sequences lack homology to the
region of interest, is facilitated by introducing into a cell,
along with the exogenous sequences, a nucleoprotein filament made
up of sequences homologous to the region of interest coated with
fusion molecules comprising a recombinase domain and a
sequence-specific DNA-binding domain. Inclusion of the
sequence-specific DNA-binding domain in the fusion protein
increases the frequency of recombination observed compared to
instances in which a nucleoprotein filament is formed using a
recombinase alone. It is not necessary that a target sequence, or
binding site, for the sequence-specific DNA-binding domain be
present in either the exogenous sequence or the genomic region of
interest.
[0089] Without wishing to be bound by any particular theory, a
possible explanation for the ability of nucleoprotein filaments
comprising the fusion proteins disclosed herein to stimulate gene
targeting is that the recombinase portion of the fusion protein
catalyzes double-stranded breaks in genomic DNA homologous to the
nucleotide sequence of the DNA component of the filament. It is
well-known that double-stranded breaks in cellular DNA stimulate
cellular repair mechanisms, by several thousand-fold, in the
vicinity of the cleavage site, facilitating both homology-dependent
(see below) and homology-independent integration of exogenous
sequences. See, for example, Rouet et al. (1994) Mol. Cell. Biol.
14:8096-8106; Choulika et al. (1995) Mol. Cell. Biol. 15:1968-1973;
Donoho et al. (1998) Mol. Cell. Biol. 18:4070-4078; Johnson et al.
(2001) Biochem. Soc. Trans. 29:196-201; and Yanez et al. (1998)
Gene Therapy 5:149-159.
[0090] Targeted non-homology-dependent integration, as described
above, can be used, e.g., for purposes of cell engineering and/or
protein overexpression. Embodiments include donor sequences that
lack homology to the host DNA and/or that lack homology to the
intended site of insertion. For instance, the donor nucleic acid
may be designed or chosen to lack homology to nucleic acids at or
near the site targeted by the probe, e.g., within 0 to 500,000 bp
of the probe; artisans will immediately appreciate that all the
ranges and values within the explicitly stated ranges are
contemplated, e.g., within about 100,000 bp. The lack of homology
can be, for example, having no more than 50% sequence identity
and/or lacking in specific hybridization at low stringency. The
lack of homology can further include a criterion of having no more
than 9 bp identity. Further criteria for non-homology may be
inferred from the following discussion of homologous recombination.
Embodiments include cells, in vitro cells, cells treated ex vivo
for reincorporation into the host animal (e.g., human), in vivo
cells, animals, transgenic animals, and synthetic DNA modified with
non-homologous donor DNA, as well as systems and methods for
producing the same as disclosed herein.
Homologous Recombination
[0091] Also described herein are methods of facilitating homologous
recombination between a chromosomal locus and an exogenous nucleic
acid bearing sequences that are homologous to the chromosomal locus
(e.g., gene targeting). Such mechanisms can result either in the
replacement of a genomic sequence (e.g., a region of interest in a
cellular genome) with a homologous non-identical sequence or in the
insertion, into a genome, of exogenous sequences not normally
present in that genome (provided that the sequences not normally
present in the genome are linked, in the exogenous nucleic acid,
with sequences that are homologous to a region of interest in the
genome). Embodiments include cells, in vitro cells, cells treated
ex vivo for reincorporation into the host animal (e.g., human), in
vivo cells, animals, transgenic animals, and synthetic DNA modified
with homologous donor DNA, as well as systems and methods for
producing the same as disclosed herein.
[0092] The disclosed methods for targeted recombination involve the
introduction, into a cell, of an exogenous nucleic acid comprising
sequences homologous to the region of interest, along with a fusion
molecule comprising a recombinase domain and a sequence-specific
DNA-binding domain. The fusion molecules have been described above
and optionally comprise a nuclear localization signal. The
exogenous nucleic acid sequence, also referred to herein as a
"donor sequence," can be introduced into the cell prior to,
concurrently with, or subsequent to, introduction of the fusion
molecule.
[0093] A "homologous, non-identical sequence" refers to a first
sequence which shares a degree of sequence identity with a second
sequence, but whose sequence is not identical to that of the second
sequence. For example, a polynucleotide comprising the wild-type
sequence of a mutant gene is homologous and non-identical to the
sequence of the mutant gene. Similarly, two alleles of a gene are
homologous, non-identical sequences, as are two haplotypes of a
particular genomic locus.
[0094] Embodiments include an exogenous, or donor, nucleic acid
that contains substantial homology, which is sufficient homology to
a genomic sequence to support homologous recombination (or
homology-directed repair) between it and the genomic sequence to
which it bears homology: approximately 25, 50 100, 200, 500, 750,
1,000, 1,500, 2,000 nucleotides or more of sequence homology
between a donor and a genomic sequence (or any integral value
between 10 and 2,000 nucleotides, or more) will generally support
homologous recombination therebetween. Donor sequences can range in
length, for example, from 10 to 10,000 nucleotides (or any integral
value of nucleotides therebetween) or longer. It will be readily
apparent that the donor sequence is typically not identical to the
genomic sequence that it replaces. For example, the sequence of the
donor polynucleotide can contain one or more single base changes,
insertions, deletions, inversions or rearrangements with respect to
the genomic sequence, so long as sufficient homology with
chromosomal sequences is present. Alternatively, a donor sequence
can contain a non-homologous sequence flanked by two regions of
homology, or a homologous sequence flanked by two regions of
non-homology. Additionally, donor sequences can comprise a vector
molecule containing sequences that are not homologous to the region
of interest in cellular chromatin. A donor molecule can contain
several, discontinuous regions of homology to cellular chromatin.
For example, for targeted insertion of sequences not normally
present in a region of interest, said sequences can be present in a
donor nucleic acid molecule and flanked by regions of homology to
sequences in the region of interest.
[0095] Generally, the homologous region(s) of a donor sequence will
have at least 50% sequence identity to a genomic sequence with
which recombination is desired. In certain embodiments, 60%, 70%,
80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present;
artisans will immediately appreciate that all the ranges and values
within the explicitly stated values are contemplated.
[0096] Thus, in certain embodiments, the degree of homology between
two sequences (e.g. a genomic locus and an exogenous nucleic acid)
is substantial to allow homologous recombination therebetween. Two
homologous non-identical sequences can be any length and their
degree of non-homology can be as small as a single nucleotide
(e.g., for correction of a genomic point mutation by targeted
homologous recombination) or as large as 10 or more kilobases
(e.g., for insertion of a gene at a predetermined ectopic site in a
chromosome). Two polynucleotides comprising the homologous
non-identical sequences need not be the same length. For example,
an exogenous polynucleotide (i.e., a donor polynucleotide) of
between 20 and 10,000 nucleotides or nucleotide pairs can be
used.
[0097] Techniques for determining nucleic acid and amino acid
sequence identity and homology are known in the art. Typically,
such techniques include determining the nucleotide sequence of the
mRNA for a gene and/or determining the amino acid sequence encoded
thereby, and comparing these sequences to a second nucleotide or
amino acid sequence. Genomic sequences can also be determined and
compared in this fashion. In general, identity refers to an exact
nucleotide-to-nucleotide or amino acid-to-amino acid correspondence
of two polynucleotides or polypeptide sequences, respectively. Two
or more sequences (polynucleotide or amino acid) can be compared by
determining their percent identity. The percent identity of two
sequences, whether nucleic acid or amino acid sequences, is the
number of exact matches between two aligned sequences divided by
the length of the shorter sequences and multiplied by 100. An
approximate alignment for nucleic acid sequences is provided by the
local homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2:482-489 (1981). This algorithm can be applied to
amino acid sequences by using the scoring matrix developed by
Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff
ed., 5 suppl. 3:353-358, National Biomedical Research Foundation,
Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res.
14(6):6745-6763 (1986). An exemplary implementation of this
algorithm to determine percent identity of a sequence is provided
by the Genetics Computer Group (Madison, Wis.) in the "BestFit"
utility application. The default parameters for this method are
described in the Wisconsin Sequence Analysis Package Program
Manual, Version 8 (1995) (available from Genetics Computer Group,
Madison, Wis.).
[0098] Another method of establishing percent identity is to use
the MPSRCH package of programs copyrighted by the University of
Edinburgh, developed by John F. Collins and Shane S. Sturrok, and
distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From
this suite of packages the Smith-Waterman algorithm can be employed
in which default parameters are used for the scoring table (for
example, gap open penalty of 12, gap extension penalty of one, and
a gap of six). From the data generated, the "Match" value reflects
sequence identity.
[0099] Other suitable programs for calculating the percent identity
or similarity between sequences are generally known in the art, for
example, another alignment program is BLAST, used with default
parameters. For example, BLASTN and BLASTP can be used using the
following default parameters: genetic code=standard; filter=none;
strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50
sequences; sort by=HIGH SCORE; Databases=non-redundant,
GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss
protein+Spupdate+PIR. Details of these programs can be found on the
World Wide Web at ncbi.nlm.gov/cgi-bin/- BLAST. With respect to
sequences described herein, the range of desired degrees of
sequence identity is approximately 80% to 100% and any integer
value therebetween. Typically the percent identities between
sequences are at least 70-75%, alternatively 80-82%, alternatively
85-90%, 92% or more, 95% or more, 98% or more, or 99% or more.
[0100] Alternatively, the degree of sequence similarity between
polynucleotides can be determined by hybridization of
polynucleotides under conditions that allow formation of stable
duplexes between homologous regions, followed by assay for
double-stranded nucleic acid (e.g., hyperchromicity, binding to
hydroxyapatite, or digestion with single-stranded-specific
nuclease(s), and size determination of the digested fragments). Two
nucleic acid, or two polypeptide sequences are substantially
homologous to each other when the sequences exhibit at least about
70%-75%, alternatively 80-82%, alternatively 85%-90%, 92% or more,
95% or more, 98% or more, or 99% or more sequence identity over a
defined length of the molecules, as determined using the methods
above. As used herein, substantially homologous also refers to
sequences showing complete identity to a specified DNA or
polypeptide sequence. DNA sequences that are substantially
homologous can be identified in a Southern hybridization experiment
under, for example, stringent conditions, as defined for that
particular system. Defining appropriate hybridization conditions is
within the skill of the art. See, e.g., Sambrook et al., supra;
Nucleic Acid Hybridization: A Practical Approach, editors B. D.
Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL
Press.
[0101] Selective hybridization of two nucleic acid fragments can be
determined as follows. The degree of sequence identity between two
nucleic acid molecules affects the efficiency and strength of
hybridization events between such molecules. A partially identical
nucleic acid sequence will at least partially inhibit the
hybridization of a completely identical sequence to a homologous or
identical target molecule. Inhibition of hybridization of the
completely identical sequence can be assessed using hybridization
assays that are well known in the art (e.g., Southern (DNA) blot,
Northern (RNA) blot, solution hybridization, or the like, see
Sambrook, et al., supra). Such assays can be conducted using
varying degrees of selectivity, for example, using conditions
varying from low to high stringency. If conditions of low
stringency are employed, the absence of non-specific binding can be
assessed using a secondary probe that lacks even a partial degree
of sequence identity (for example, a probe having less than about
30% sequence identity with the target molecule), such that, in the
absence of non-specific binding events, the secondary probe will
not hybridize to the target.
[0102] When utilizing a hybridization-based detection system, a
nucleic acid probe is chosen that is complementary to a reference
nucleic acid sequence, and then, by selection of appropriate
conditions, the probe and the reference sequence selectively
hybridize, or anneal, to each other to form a duplex molecule. A
nucleic acid molecule that is capable of hybridizing selectively to
a reference sequence under moderately stringent hybridization
conditions typically hybridizes under conditions that allow
detection of a target nucleic acid sequence of at least about 10-14
nucleotides in length having at least approximately 70% sequence
identity with the sequence of the selected nucleic acid probe.
Stringent hybridization conditions typically allow detection of
target nucleic acid sequences of at least about 10-14 nucleotides
in length having a sequence identity of greater than about 90-95%
with the sequence of the selected nucleic acid probe. Hybridization
conditions useful for probe/reference sequence hybridization, where
the probe and reference sequence have a specific degree of sequence
identity, can be determined as is known in the art (see, for
example, Nucleic Acid Hybridization: A Practical Approach, editors
B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL
Press).
[0103] Conditions for hybridization are well-known to those of
skill in the art. Hybridization stringency refers to the degree to
which hybridization conditions disfavor the formation of hybrids
containing mismatched nucleotides, with higher stringency
correlated with a lower tolerance for mismatched hybrids. Factors
that affect the stringency of hybridization are well-known to those
of skill in the art and include, but are not limited to,
temperature, pH, ionic strength, and concentration of organic
solvents such as, for example, formamide and dimethylsulfoxide. As
is known to those of skill in the art, hybridization stringency is
increased by higher temperatures, lower ionic strength and lower
solvent concentrations.
[0104] With respect to stringency conditions for hybridization, it
is well known in the art that numerous equivalent conditions can be
employed to establish a particular stringency by varying, for
example, the following factors: the length and nature of the
sequences, base composition of the various sequences,
concentrations of salts and other hybridization solution
components, the presence or absence of blocking agents in the
hybridization solutions (e.g., dextran sulfate, polyethylene
glycol), hybridization reaction temperature and time parameters and
wash conditions.
[0105] The exogenous, donor polynucleotide can be DNA or RNA,
single-stranded or double-stranded and can be introduced into a
cell in linear or circular form. If introduced in linear form, the
ends of the donor sequence can be protected (e.g., from
exonucleolytic degradation) by methods known to those of skill in
the art. For example, one or more dideoxynucleotide residues are
added to the 3' terminus of a linear molecule and/or
self-complementary oligonucleotides are ligated to one or both
ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci.
USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889.
Additional methods for protecting exogenous polynucleotides from
degradation include, but are not limited to, addition of terminal
amino group(s) and the use of modified internucleotide linkages
such as, for example, phosphorothioates, phosphoramidates, and
O-methyl ribose or deoxyribose residues. A polynucleotide can be
introduced into a cell as part of a vector molecule having
additional sequences such as, for example, replication origins,
promoters and genes encoding antibiotic resistance. Moreover, donor
polynucleotides can be introduced as naked nucleic acid, as nucleic
acid complexed with an agent such as a liposome or poloxamer, or
can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus,
retrovirus, lentivirus).
[0106] In additional embodiments, the ends of an exogenous donor
nucleic acid molecule can be modified in ways that make it a more
suitable substrate for recombination. For example, an exogenous
nucleic acid molecule for integration into a genome, by either a
homology-dependent or a non-homology-dependent mechanism, can
contain 3'-protruding single-stranded ends ("3' overhangs").
Methods for generating such ends (e.g., treating linear
double-stranded DNA with 5'-specific exonucleases, such as .lamda.
exonuclease or T7 exonuclease) are known in the art.
Ancillary Methods for Enhancing Recombination Frequency
[0107] Methods and compositions are also provided that enhance
levels of targeted recombination including, but not limited to, the
use of cDNAs and/or engineered transcription factors to increase
expression of genes involved in homologous recombination, such as,
for example, members of the RAD52 epistasis group (e.g., Rad50,
Rad51, Rad51B, Rad51C, Rad51D, Rad52, Rad54, Rad54B, Mre11, XRCC2,
XRCC3), genes whose products interact with the aforementioned gene
products (e.g., BRCA1, BRCA2) and/or genes in the NBS1 complex.
When homologous recombination is desired, similar methods can be
used, in combination with the methods and compositions disclosed
herein, to repress expression of genes involved in non-homologous
end joining (e.g., Ku70/80, XRCC4, poly(ADP ribose) polymerase, DNA
ligase 4). See, for example, Yanez et al. (1998) Gene Therapy
5:149-159; Hoeijmakers (2001) Nature 411:366-374; Johnson et al.
(2001) Biochem. Soc. Trans. 29:196-201; Tauchi et al. (2002)
Oncogene 21:8967-8980. Methods for activation and repression of
gene expression using fusions between a zinc finger binding domain
and a functional domain are disclosed, for example, in U.S. Pat.
Nos. 6,534,261; 6,824,978 and 6,933,113. Additional repression
methods include the use of antisense oligonucleotides and/or small
interfering RNA (siRNA or RNAi) targeted to the sequence of the
gene to be repressed.
[0108] Additional proteins involved in gene conversion and
recombination-related chromatin remodeling, which can be used in
the aforementioned methods and compositions, include histone
acetyltransferases (e.g., Esa1p, Tip60), histone methyltransferases
(e.g., Dot1p), histone kinases and histone phosphatases.
[0109] The p53 protein has been reported to play a central role in
repressing homologous recombination. See, for example, Valerie et
al., (2003) Oncogene 22:5792-5812; Janz, et al. (2002) Oncogene
21:5929-5933. For example, the rate of homologous recombination in
p53-deficient human tumor lines is 10,000-fold greater than in
primary human fibroblasts, and there is a 100-fold increase in
homologous recombination in tumor cells with a non-functional p53,
compared to those with functional p53. Mekeel et al. (1997)
Oncogene 14:1847-1857. In addition, overexpression of p53
dominant-negative mutants leads to a 20-fold increase in
spontaneous recombination. Bertrand et al. (1997) Oncogene
14:1117-1122. Analysis of different p53 mutations has revealed that
the roles of p53 in transcriptional transactivation and G1 cell
cycle checkpoint control are separable from its involvement in
homologous recombination. Saintigny et al. (1999) Oncogene
18:3553-3563; Boehden et al. (2003) Oncogene 22:4111-4117.
Accordingly, downregulation of p53 activity can serve to increase
the efficiency of targeted homologous recombination using the
methods and compositions disclosed herein. Any method for
downregulation of p53 activity can be used, including but not
limited to cotransfection and overexpression of a p53 dominant
negative mutant or targeted repression of p53 gene expression
according to methods disclosed, e.g., in U.S. Pat. No.
6,534,261.
[0110] Further increases in efficiency of targeted recombination
can be achieved by blocking the cells in the G.sub.2 phase of the
cell cycle, when homology-driven repair processes are maximally
active. Such arrest can be achieved in a number of ways. For
example, cells can be treated with e.g., drugs, compounds and/or
small molecules which influence cell-cycle progression so as to
arrest cells in G.sub.2 phase. Exemplary molecules of this type
include, but are not limited to, compounds which affect microtubule
polymerization (e.g., vinblastine, nocodazole, Taxol), compounds
that interact with DNA (e.g., cis-platinum(II) diamine dichloride,
Cisplatin, doxorubicin) and/or compounds that affect DNA synthesis
(e.g., thymidine, hydroxyurea, L-mimosine, etoposide,
5-fluorouracil). Additional increases in recombination efficiency
are achieved by the use of histone deacetylase (HDAC) inhibitors
(e.g., sodium butyrate, trichostatin A) which alter chromatin
structure to make genomic DNA more accessible to the cellular
recombination machinery.
[0111] Additional methods for cell-cycle arrest include
overexpression of proteins which inhibit the activity of the CDK
cell-cycle kinases, for example, by introducing a cDNA encoding
such a protein into the cell or by activating expression of the
gene encoding the protein in the cell. Cell-cycle arrest is also
achieved by inhibiting the activity of cyclins and CDKs, for
example, using RNAi methods (e.g., U.S. Pat. No. 6,506,559) or by
inhibiting the expression of one or more genes involved in
cell-cycle progression such as, for example, cyclin and/or CDK.
[0112] Targeted homology-dependent integration, as described above,
can be used, e.g., for purposes of cell engineering and/or protein
overexpression or to replace a wild-type sequence with a mutant
sequence (or vice versa).
Targeted Mutagenesis
[0113] Any of the methods disclosed herein can be used for targeted
mutagenesis by, for example, insertion of a sequence into a gene so
as to disrupt the gene, introduction of a deletion, introduction of
a point mutation or replacement of a gene by a non-functional
allele. Such targeted mutagenesis can be used for a number of
purposes. For example, targeted mutagenesis of genes encoding viral
receptors (e.g., the CCR5 and CXCR4 receptors for HIV) can be used
to render the receptors unable to bind to virus, thereby preventing
new infection and blocking the spread of existing infections.
Non-limiting examples of viruses or viral receptors that may be
targeted include herpes simplex virus (HSV), such as HSV-1 and
HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV) and
cytomegalovirus (CMV), HHV6 and HHV7. The hepatitis family of
viruses includes hepatitis A virus (HAV), hepatitis B virus (HBV),
hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis
E virus (HEV) and hepatitis G virus (HGV). Other viruses or their
receptors can also be targeted, including, but not limited to,
Picomaviridae (e.g., polioviruses, etc.); Caliciviridae;
Togaviridae (e.g., rubella virus, dengue virus, etc.);
Flaviviridae; Coronaviridae; Reoviridae; Bimaviridae;
Rhabodoviridae (e.g., rabies virus, etc.); Filoviridae;
Paramyxoviridae (e.g., mumps virus, measles virus, respiratory
syncytial virus, etc.); Orthomyxoviridae (e.g., influenza virus
types A, B and C, etc.); Bunyaviridae; Arenaviridae; Retroviradae;
lentiviruses (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III,
LAV, ARV, hTLR, etc.) HIV-II); simian immunodeficiency virus (SIV),
human papillomaviruses (HPVs), and the tick-borne encephalitis
viruses. See, e.g. Virology, 3rd Edition (W. K. Joklik ed. 1988);
Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe,
eds. 1991), for a description of these and other viruses.
[0114] In similar fashion, the genome of an infecting bacterium can
be mutagenized by one or more of the methods disclosed herein, to
block or ameliorate bacterial infections.
[0115] Targeted DNA cleavage and targeted recombination, as
disclosed herein, can be used to alter non-coding sequences (e.g.,
regulatory sequences such as promoters, enhancers, initiators,
terminators, splice sites) to alter the levels of expression of a
gene product. Such methods can be used, for example, for
therapeutic purposes, functional genomics and/or target validation
studies.
[0116] In additional embodiments utilizing the compositions and
methods described herein, genes encoding HLA proteins involved in
graft rejection can be cleaved, mutagenized or altered by
recombination, in either their coding or regulatory sequences, so
that their expression is blocked or they express a non-functional
product. For example, by inactivating the gene encoding the common
beta subunit gene (beta2-microglobulin), HLA class I null stem
cells can be generated from any donor, thereby reducing the need
for closely matched donor/recipient MHC haplotypes during stem cell
grafting.
Genetic Diseases
[0117] The disclosed methods for targeted recombination (both
homology-dependent and non-homology-dependent) can be used to
replace any genomic sequence with a homologous, non-identical
sequence. For example, a mutant genomic sequence can be replaced by
its wild-type counterpart, thereby providing methods for treatment
of e.g., genetic disease, inherited disorders, cancer, and
autoimmune disease. In like fashion, one allele of a gene can be
replaced by a different allele using the methods of targeted
recombination disclosed herein.
[0118] Exemplary genetic diseases include, but are not limited to,
achondroplasia, achromatopsia, acid maltase deficiency, acquired
immunodeficiencies, adenosine deaminase deficiency (OMIM No.
102700), adrenoleukodystrophy, aicardi syndrome, alpha-I
antitrypsin deficiency, alpha-thalassemia, androgen insensitivity
syndrome, apert syndrome, arrhythmogenic right ventricular,
dysplasia, ataxia telangictasia, barth syndrome, beta-thalassemia,
blue rubber bleb nevus syndrome, canavan disease, chronic
granulomatous diseases (CGD), cri du chat syndrome, cystic
fibrosis, dercum's disease, ectodermal dysplasia, Fanconi's anemia,
fibrodysplasia ossificans progressive, fragile X syndrome,
galactosemis, Gaucher's disease, generalized gangliosidoses (e.g.,
GM1), hemochromatosis, hemoglobinopathies (e.g., sickle cell
anemia, the hemoglobin C mutation in the 6.sup.th codon of
beta-globin, alpha-thalassemia, beta-thalassemia), hemophilia,
Huntington's disease, Hurler Syndrome, hypophosphatasia,
Klinefleter syndrome, Krabbes Disease, Langer-Giedion Syndrome,
leukocyte adhesion deficiency (LAD, OMIM No. 116920),
leukodystrophy, long QT syndrome, lysosomal storage diseases (e.g.,
Gaucher's disease, GM1, Fabry disease and Tay-Sachs disease),
Marfan syndrome, Moebius syndrome, mucopolysaccahidosis (e.g.
Hunter's disease, Hurler's disease), nail patella syndrome,
nephrogenic diabetes insipdius, neurofibromatosis, Neimann-Pick
disease, osteogenesis imperfecta, porphyria, Prader-Willi syndrome,
progeria, Proteus syndrome, retinoblastoma, Rett syndrome,
Rubinstein-Taybi syndrome, Sanfilippo syndrome, severe combined
immunodeficiency (SCID), Shwachman syndrome, sickle cell disease
(sickle cell anemia), Smith-Magenis syndrome, Stickler syndrome,
Tay-Sachs disease, Thrombocytopenia Absent Radius (TAR) syndrome,
Treacher Collins syndrome, trisomy, tuberous sclerosis, Turner's
syndrome, urea cycle disorder, von Hippel-Landau disease,
Waardenburg syndrome, Williams syndrome, Wilson's disease,
Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome
(XLP, OMIM No. 308240).
[0119] In many of these cases, a region of interest comprises a
mutation, and the exogenous, or donor nucleic acid comprises the
corresponding wild-type sequence. Similarly, a wild-type genomic
sequence can be replaced by a mutant sequence, if such is
desirable. For example, overexpression of an oncogene can be
reversed either by mutating the gene or by replacing its control
sequences with sequences that support a lower, non-pathologic level
of expression. As another example, the wild-type allele of the
ApoAI gene can be replaced by the ApoAI Milano allele, to treat
atherosclerosis. Indeed, any pathology dependent upon a particular
genomic sequence, in any fashion, can be corrected or alleviated
using the methods and compositions disclosed herein.
[0120] In certain cases, alteration of a genomic sequence in a
pluripotent cell (e.g., a hematopoietic stem cell) is desired.
Methods for mobilization, enrichment and culture of hematopoietic
stem cells are known in the art. See for example, U.S. Pat. Nos.
5,061,620; 5,681,559; 6,335,195; 6,645,489 and 6,667,064. Treated
stem cells can be returned to a patient for treatment of various
diseases including, but not limited to, SCID and sickle-cell
anemia.
[0121] The genome of totipotent stem cells can also be altered by
the use of the methods and compositions disclosed herein.
Totipotent stem cells are described, for example, in U.S. Pat. Nos.
5,843,780; 6,200,806 and 7,029,913. Totipotent stem cells can be
cultured (e.g., U.S. Pat. Nos. 6,602,711 and 7,005,252) and
differentiated into various types of pluripotent cells (e.g., U.S.
Pat. Nos. 6,280,718; 6,613,568 and 6,887,706), which can also be
used in the practice of the disclosed methods.
[0122] Similarly, the genomes of induced pluripotent stem cells
(iPS cells) can also be modified according to the disclosed methods
and compositions. Induced pluripotent stem cells are described, for
example, in Yu et al. (2007) Science 318: 1917-1920 and Dimos et
al. (2008) Science 321:1218-1221.
[0123] Accordingly, embodiments of the invention include direction
of a proteinaceous fusion molecule, a probe, and an exogenous
nucleic acid to an animal (includes human or non-human, mammalian,
and vertebrate) to treat the animal. The exogenous nucleic acid
expresses a protein that provides a therapeutic effect. In other
cases, the offending genetic basis for the disease is deleted. The
method may be performed without a vector, e.g., without a virus,
and without a transposon.
Transgenic Animals
[0124] The disclosed methods and compositions can be used for
generation of transgenic livestock and large mammals, as disclosed,
for example, in U.S. Pat. No. 7,199,218, and U.S. Ser. No.
12/504,364 filed Jul. 16, 2009 (U.S. Pub. No 2010/0146655, Methods
And Materials For Producing Transgenic Animals) the disclosures of
which are hereby incorporated herein by reference for all purposes
including the purposes of describing methods for making transgenic
animals, methods for targeted genome alteration, and uses of
transgenic animals. In all cases, the present specification
controls in case of conflict with documents incorporated by
reference.
[0125] Transgenic artiodactyls can be made (e.g., pigs, sheep,
goats, and cows). The nucleated cells of the transgenic
artiodactyls provided herein contain a nucleic acid construct
described herein. As used herein, "transgenic artiodactyl" includes
founder transgenic artiodactyls as well as progeny of the founders,
progeny of the progeny, and so forth, provided that the progeny
retain the nucleic acid construct. For example, a transgenic
founder animal can be used to breed additional animals that contain
the nucleic acid construct.
[0126] Tissues obtained from the transgenic artiodactyls (e.g.,
transgenic pigs) and cells derived from the transgenic artiodactyls
(e.g., transgenic pigs) also are provided herein. As used herein,
"derived from" indicates that the cells can be isolated directly
from the animal or can be progeny of such cells. For example,
brain, lung, liver, pancreas, heart and heart valves, muscle,
kidney, thyroid, corneal, skin, blood vessels or other connective
tissue can be obtained from a transgenic artiodactyl (e.g.,
transgenic pig). Blood and hematopoietic cells, Islets of
Langerhans, beta cells, brain cells, hepatocytes, kidney cells, and
cells from other organs and body fluids, for example, also can be
derived from transgenic artiodactyls (e.g., transgenic pigs).
Organs and cells from transgenic pigs can be transplanted into a
human patient. For example, islets from transgenic pigs can be
transplanted to human diabetic patients.
[0127] Various techniques known in the art can be used to introduce
nucleic acid constructs into non-human animals to produce founder
lines, in which the nucleic acid construct is integrated into the
genome. Such techniques include, without limitation, pronuclear
microinjection (U.S. Pat. No. 4,873,191), retrovirus mediated gene
transfer into germ lines (Van der Putten et al. (1985) Proc. Natl.
Acad. Sci. USA 82, 6148-1652), gene targeting into embryonic stem
cells (Thompson et al. (1989) Cell 56, 313-321), electroporation of
embryos (Lo (1983) Mol. Cell. Biol. 3, 1803-1814), sperm mediated
gene transfer (Lavitrano et al. (2002) Proc. Natl. Acad. Sci. USA
99, 14230-14235; Lavitrano et al. (2006) Reprod. Fert. Develop. 18,
19-23), and in vitro transformation of somatic cells, such as
cumulus or mammary cells, or adult, fetal, or embryonic stem cells,
followed by nuclear transplantation (Wilmut et al. (1997) Nature
385, 810-813; and Wakayama et al. (1998) Nature 394, 369-374).
Pronuclear microinjection, sperm mediated gene transfer, and
somatic cell nuclear transfer are particularly useful
techniques.
[0128] Typically, in pronuclear microinjection, a nucleic acid
construct described herein is introduced into a fertilized egg; 1
or 2 cell fertilized eggs are used as the pronuclei containing the
genetic material from the sperm head and the egg are visible within
the protoplasm. Pronuclear staged fertilized eggs can be obtained
in vitro or in vivo (i.e., surgically recovered from the oviduct of
donor animals). In vitro fertilized eggs can be produced as
follows. For example, swine ovaries can be collected at an
abattoir, and maintained at 22-28.degree. C. during transport.
Ovaries can be washed and isolated for follicular aspiration, and
follicles ranging from 4-8 mm can be aspirated into 50 mL conical
centrifuge tubes using 18 gauge needles and under vacuum.
Follicular fluid and aspirated oocytes can be rinsed through
pre-filters with commercial TL-HEPES (Minitube, Verona, Wis.).
Oocytes surrounded by a compact cumulus mass can be selected and
placed into TCM-199 Oocyte Maturation Medium (Minitube, Verona,
Wis.) supplemented with 0.1 mg/mL cysteine, 10 ng/mL epidermal
growth factor, 10% porcine follicular fluid, 50 .mu.M
2-mercaptoethanol, 0.5 mg/ml cAMP, 10 IU/mL each of pregnant mare
serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG)
for approximately 22 hours in humidified air at 38.7.degree. C. and
5% CO.sub.2. Subsequently, the oocytes can be moved to fresh
TCM-199 maturation medium which will not contain cAMP, PMSG or hCG
and incubated for an additional 22 hours. Matured oocytes can be
stripped of their cumulus cells by vortexing in 0.1% hyaluronidase
for 1 minute.
[0129] Mature oocytes can be fertilized in 500 .mu.l MINITUBE
PORCPRO IVF MEDIUM SYSTEM (Minitube, Verona, Wis.) in Minitube
5-well fertilization dishes. In preparation for in vitro
fertilization (IVF), freshly-collected or frozen boar semen can be
washed and resuspended in PORCPRO IVF Medium to 4.times.10.sup.5
sperm. Sperm concentrations can be analyzed by computer assisted
semen analysis (SPERMVISION, Minitube, Verona, Wis.). Final in
vitro insemination can be performed in a 10 .mu.l volume at a final
concentration of approximately 40 motile sperm/oocyte, depending on
boar. Incubate all fertilizing oocytes at 38.7.degree. C. in 5.0%
CO.sub.2 atmosphere for 6 hours. Six hours post-insemination,
presumptive zygotes can be washed twice in NCSU-23 and moved to 0.5
mL of the same medium. This system can produce 20-30% blastocysts
routinely across most boars with a 10-30% polyspermic insemination
rate.
[0130] Linearized nucleic acid constructs can be injected into one
of the pronuclei then the injected eggs can be transferred to a
recipient female (e.g., into the oviducts of a recipient female)
and allowed to develop in the recipient female to produce the
transgenic animals. In particular, in vitro fertilized embryos can
be centrifuged at 15,000.times.g for 5 minutes to sediment lipids
allowing visualization of the pronucleus. The embryos can be
injected with approximately 5 picoliters of the
transposon/transposase cocktail using an Eppendorf FEMTOJET
injector and can be cultured until blastocyst formation (.about.144
hours) in NCSU 23 medium (see, e.g., WO/2006/036975). Rates of
embryo cleavage and blastocyst formation and quality can be
recorded.
[0131] Embryos can be surgically transferred into uteri of
asynchronous recipients. For surgical embryo transfer, anesthesia
can be induced with a combination of the following: ketamine (2
mg/kg); tiletamine/zolazepam (0.25 mg/kg); xylazine (1 mg/kg); and
atropine (0.03 mg/kg) (all from Columbus Serum). While in dorsal
recumbency, the recipients can be aseptically prepared for surgery
and a caudal ventral incision can be made to expose and examine the
reproductive tract. Typically, 100-200 (e.g., 150-200) embryos can
be deposited into the ampulla-isthmus junction of the oviduct using
a 5.5-inch TOMCAT.RTM. catheter. After surgery, real-time
ultrasound examination of pregnancy can be performed using an ALOKA
900 ULTRASOUND SCANNER (Aloka Co. Ltd, Wallingford, Conn.) with an
attached 3.5 MHz trans-abdominal probe. Monitoring for pregnancy
initiation can begin at 23 days post fusion and can be repeated
weekly during pregnancy. Recipient husbandry can be maintained as
normal gestating sows.
[0132] In somatic cell nuclear transfer, a transgenic artiodactyl
cell (e.g., a transgenic pig cell) such as an embryonic blastomere,
fetal fibroblast, adult ear fibroblast, or granulosa cell that
includes a nucleic acid construct described above, can be
introduced into an enucleated oocyte to establish a combined cell.
Oocytes can be enucleated by partial zona dissection near the polar
body and then pressing out cytoplasm at the dissection area.
Typically, an injection pipette with a sharp beveled tip is used to
inject the transgenic cell into an enucleated oocyte arrested at
meiosis 2. In some conventions, oocytes arrested at meiosis 2 are
termed "eggs." After producing a porcine embryo (e.g., by fusing
and activating the oocyte), the porcine embryo is transferred to
the oviducts of a recipient female, about 20 to 24 hours after
activation. See, for example, Cibelli et al. (1998) Science 280,
1256-1258 and U.S. Pat. No. 6,548,741. For pigs, recipient females
can be checked for pregnancy approximately 20-21 days after
transfer of the embryos.
[0133] Standard breeding techniques can be used to create animals
that are homozygous for the target nucleic acid from the initial
heterozygous founder animals. Homozygosity may not be required,
however. Transgenic pigs described herein can be bred with other
pigs of interest.
[0134] Once transgenic animals have been generated, expression of a
target nucleic acid can be assessed using standard techniques.
Initial screening can be accomplished by Southern blot analysis to
determine whether or not integration of the construct has taken
place. For a description of Southern analysis, see sections
9.37-9.52 of Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, second edition, Cold Spring Harbor Press, Plainview; NY.
Polymerase chain reaction (PCR) techniques also can be used in the
initial screening. PCR refers to a procedure or technique in which
target nucleic acids are amplified. Generally, sequence information
from the ends of the region of interest or beyond is employed to
design oligonucleotide primers that are identical or similar in
sequence to opposite strands of the template to be amplified. PCR
can be used to amplify specific sequences from DNA as well as RNA,
including sequences from total genomic DNA or total cellular RNA.
Primers typically are 14 to 40 nucleotides in length, but can range
from 10 nucleotides to hundreds of nucleotides in length. PCR is
described in, for example PCR Primer: A Laboratory Manual, ed.
Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press,
1995. Nucleic acids also can be amplified by ligase chain reaction,
strand displacement amplification, self-sustained sequence
replication, or nucleic acid sequence-based amplified. See, for
example, Lewis (1992) Genetic Engineering News 12, 1; Guatelli et
al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874-1878; and Weiss
(1991) Science 254, 1292-1293. At the blastocyst stage, embryos can
be individually processed for analysis by PCR, Southern
hybridization and splinkerette PCR (see, e.g., Dupuy et al. Proc
Natl Acad Sci USA (2002) 99(7):4495-4499).
[0135] Expression of a nucleic acid sequence encoding a polypeptide
in the tissues of transgenic pigs can be assessed using techniques
that include, without limitation, Northern blot analysis of tissue
samples obtained from the animal, in situ hybridization analysis,
Western analysis, immunoassays such as enzyme-linked immunosorbent
assays, and reverse-transcriptase PCR (RT-PCR).
Administration
[0136] The fusion molecules, nucleoproteins and/or nucleic acids
disclosed herein can be administered directly to a subject for
therapeutic or prophylactic applications such as those described
herein. Subjects can be animals or plants. In particular, plant
subjects can be monocotyledonous or dicotyledonous. Animal subjects
can be vertebrates, in particular mammals (e.g., livestock, pets),
in particular primates, in particular humans.
[0137] In general, and in view of the discussion herein, reference
to the introduction of a fusion protein into a subject can mean
either that a fusion protein itself is introduced or that a nucleic
acid encoding a fusion protein is introduced in a form that can be
expressed in the subject.
[0138] With respect to the introduction of nucleic acids and
nucleoprotein filaments into cells, any of the well-known
procedures for introducing nucleic acids into cells can also be
used for introduction of nucleoprotein filaments. For example,
methods of non-viral delivery of nucleic acids and nucleoprotein
filaments include, but are not limited to, electroporation,
lipofection, microinjection, biolistics, virosomes, liposomes,
immunoliposomes, polycation or lipid:nucleic acid conjugates,
polybrene, protoplast fusion, calcium phosphate-mediated
transfection, DEAE-dextran-mediated transfection, naked DNA,
artificial virions, and agent-enhanced uptake of DNA. Lipofection
is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and
4,897,355) and lipofection reagents are available commercially
(e.g., Transfectam.TM., Lipofectamine.RTM. and Lipofectin.TM.).
Cationic and neutral lipids that are suitable for efficient
receptor-recognition lipofection of polynucleotides include those
of Felgner, WO 91/17424 and WO 91/16024. Delivery can be to cells
(in vitro or ex vivo administration) or target tissues (in vivo
administration). See also Sambrook et al., supra and Ausubel et
al., supra.
[0139] In the case of introduction of a fusion protein and an
exogenous nucleic acid, the exogenous nucleic acid may be present
in molar excess, as measured by the moles of fusion protein and
moles of DNA strands of exogenous nucleic acid. For instance, the
exogenous DNA fragment may be present in a molar concentration that
exceeds the molar concentration of the fusion protein, with the
excess optionally being at least 2-fold or between about 2-fold and
500-fold; artisans will immediately appreciate that all ranges and
values between the explicitly stated values are contemplated, e.g.,
10-fold or from about 5-fold to about 50-fold.
Expression Vectors
[0140] Nucleic acids can be cloned into various types of vectors
for transformation into prokaryotic or eukaryotic cells for
replication and/or expression, as is known in the art.
[0141] To obtain expression of, for example, a fusion protein, a
nucleic acid encoding the fusion protein can be inserted into an
expression vector that contains a promoter to direct transcription.
Suitable bacterial and eukaryotic promoters are well known in the
art and described, e.g., in Sambrook et al., Molecular Cloning, A
Laboratory Manual (2nd ed. 1989; 3.sup.rd ed., 2001); Kriegler,
Gene Transfer and Expression: A Laboratory Manual (1990); and
Ausubel et al., supra. Bacterial expression systems are available
in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.
(1983) Gene 22:229-235). Kits for such expression systems are
commercially available. Eukaryotic expression systems for mammalian
cells, yeast, and insect cells are well known in the art and are
also commercially available.
[0142] Nucleic acids can be incorporated into vectors. Vectors most
often contain one or more expression cassettes that comprise one or
more expression control sequences, wherein an expression control
sequence is a DNA sequence that controls and regulates the
transcription and/or translation of another DNA sequence or mRNA,
respectively. Expression control sequences include, for example,
promoter sequences, transcriptional enhancer elements, start
codons, stop codons, and any other nucleic acid elements required
for RNA polymerase binding, initiation, or termination of
transcription. A wide range of expression control sequences is well
known in the art and is commercially available. A transcriptional
unit in a vector may thus comprise an expression control sequence
operably linked to an exogenous nucleic acid sequence. For example,
a DNA sequence is operably linked to an expression-control
sequence, such as a promoter when the expression control sequence
controls and regulates the transcription and translation of that
DNA sequence. Examples of vectors include: plasmids (which may also
be a carrier of another type of vector), adenovirus,
adeno-associated virus (AAV), lentivirus (e.g., modified HIV-1, SIV
or FIV), retrovirus (e.g., ASV, ALV or MoMLV), and transposons
(e.g., Sleeping Beauty, P-elements, Tol-2, Frog Prince,
piggyBac).
Administration, Carriers, Pharmaceutical Compositions
[0143] Administration of therapeutically effective amounts of the
compositions disclosed herein is by any of the routes normally used
for introducing macromolecules into ultimate contact with the
tissue to be treated. Fusion molecules, or their encoding nucleic
acids, or nucleoprotein filaments, are administered in any suitable
manner, optionally in formulation with pharmaceutically acceptable
carriers. Multiple compositions can be administered concurrently or
separately by the same or different routes. Suitable methods for
administering such compositions are available and are well-known to
those of skill in the art, and, although more than one route can be
used to administer a particular composition, a particular route can
often provide a more immediate and more effective reaction than
another route.
[0144] Pharmaceutical compositions are determined in part by the
particular substance being administered, as well as by the
particular method used to administer the substance. Accordingly,
there are a wide variety of suitable formulations of pharmaceutical
compositions. See, e.g., Remington's Pharmaceutical Sciences, 17th
ed. 1985; Brunton et al., "Goodman and Gilman's The Pharmacological
Basis of Therapeutics," McGraw-Hill, 2005; University of the
Sciences in Philadelphia (eds.), "Remington: The Science and
Practice of Pharmacy," Lippincott Williams & Wilkins, 2005; and
University of the Sciences in Philadelphia (eds.), "Remington: The
Principles of Pharmacy Practice," Lippincott Williams &
Wilkins, 2008.
[0145] The pharmaceutical compositions of the present disclosure
can be made into aerosol formulations (i.e., they can be
"nebulized") to be administered via inhalation. Aerosol
formulations can be placed into pressurized acceptable propellants,
such as dichlorodifluoromethane, propane, nitrogen, and the
like.
[0146] Formulations suitable for parenteral administration, such
as, for example, by intravenous, intramuscular, intradermal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic
sterile injection solutions, which can contain antioxidants,
buffers, bacteriostats, and solutes that render the formulation
isotonic with the blood of the intended recipient, and aqueous and
non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives. In
the practice of the disclosed methods, compositions can be
administered, for example, by intravenous infusion, orally,
topically, intraperitoneally, intravesically or intrathecally.
Formulations can be presented in unit-dose or multi-dose sealed
containers, such as ampoules and vials. Administration can be
accomplished via single or divided doses. Injection solutions and
suspensions can be prepared from sterile lyophilates, powders,
granules, and tablets.
[0147] Appropriate dosages will depend upon the desired effect, the
size and/or weight of the subject, and the general health of the
subject, and can be determined by dose escalation over several
treatment sessions. The size of the dose can also be influenced by
the existence, nature, and extent of any adverse side-effects that
accompany administration.
[0148] The disclosed therapeutic compositions can include
pharmaceutically acceptable materials, compositions or vehicles,
such as a liquid or solid filler, diluent, excipient, solvent or
encapsulating material, i.e., carriers. These carriers are involved
in transporting the subject chemical from one organ, or region of
the body, to another organ, or region of the body. Each carrier
should be "acceptable" in the sense of being compatible with the
other ingredients of the formulation and not injurious to the
patient. Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: sugars, such as
lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations. Wetting agents, emulsifiers and
lubricants, such as sodium lauryl sulfate and magnesium stearate,
as well as coloring agents, release agents, coating agents,
sweetening, flavoring and perfuming agents, preservatives and
antioxidants can also be present in therapeutic compositions.
Administration into Plants
[0149] For delivery of polynucleotides and nucleoproteins into
plant cells, nucleic acids can be cloned into intermediate vectors
for transformation into prokaryotic or eukaryotic (e.g., plant)
cells for replication and/or expression. Intermediate vectors for
storage or manipulation of the nucleic acid or production of
protein can be prokaryotic vectors, (e.g., plasmids), shuttle
vectors, insect vectors, or viral vectors for example. Nucleic
acids can also cloned into an expression vector, for administration
to a bacterial cell, fungal cell, protozoal cell, or plant
cell.
[0150] Plant expression vectors and reporter genes are generally
known in the art. See, e.g., Gruber et al. (1993) in Methods of
Plant Molecular Biology and Biotechnology, Bernard R. Glick and
John E. Thompson, eds., CRC Press, Boca Raton, Fla. Such systems
include in vitro and in vivo recombinant DNA techniques, and can
utilize any other synthetic or natural recombination method. See,
e.g., Transgenic Plants: A Production System for Industrial and
Pharmaceutical Proteins, Owen and Pen eds., John Wiley & Sons,
1996; Transgenic Plants, Galun and Breiman eds., Imperial College
Press, 1997; and Applied Plant Biotechnology, Chopra, Malik, and
Bhat eds., Science Publishers, Inc., 1999.
[0151] The promoter used to direct expression of the nucleic acid
of choice depends on the particular application. For example, a
strong constitutive promoter is typically used for expression and
purification. In contrast, when a protein is to be used in vivo,
either a constitutive or an inducible promoter can be used,
depending on the particular function of the encoded protein. In
addition, a weak promoter can be used, when low but sustained
levels of protein are required. The promoter typically can also
include elements that are responsive to transactivation, e.g.,
hypoxia response elements and small molecule control systems such
as tet-regulated systems and the RU-486 system. See, e.g., Gossen
et al. (1992) Proc. Natl. Acad. Sci USA 89:5547-5551; Oligino et
al. (1998) Gene Ther. 5:491-496; Wang et al. (1997) Gene Ther.
4:432-441; Neering et al. (1996) Blood 88:1147-1155; and Rendahl et
al. (1998) Nature Biotechnol. 16:757-761.
[0152] Promoters suitable for use in plant expression systems
include, but are not limited to, viral promoters such as the 35S
RNA and 19S RNA promoters of cauliflower mosaic virus (CaMV)
(Brisson et al. (1984) Nature 310:511-514) and the coat protein
promoter of tobacco mosaic virus (TMV) (Takamatsu et al. (1987)
EMBO J. 6:307-311); plant promoters such as the promoter for the
gene encoding the small subunit of ribulose-1,5-bis-phosphate
carboxylase (RUBISCO) (Coruzzi et al. (1984) EMBO J. 3:1671-1680;
Broglie et al. (1984) Science 224:838-843; and plant heat shock
promoters, e.g. soybean hsp17.5-E or hsp17.3-B (Gurley et al.
(1986) Cell. Biol. 6:559-565). Other examples of promoters that can
be used for expression in plant cells include promoters from
tumor-inducing plasmids of Agrobacterium tumefaciens, such as the
nopaline synthase (NOS) and octopine synthase promoters; bacterial
T-DNA promoters such as mas and ocs promoters; or the figwort
mosaic virus 35S promoter.
[0153] In certain embodiments, the cauliflower mosaic virus (CaMV)
35S promoter is used. The caulimorvirus family has provided a
number of exemplary promoters for transgene expression in plants,
in particular, the (CaMV) 35S promoter. See, e.g., Kay et al.
(1987) Science 236:1299. Additional promoters from this family such
as the figwort mosaic virus promoter, the Commelina yellow mottle
virus promoter, and the rice tungro bacilliform virus promoter have
been described in the art, and can also be used in the methods and
compositions disclosed herein. See, e.g., Sanger et al. (1990)
Plant Mol. Biol. 14:433-443; Medberry et al. (1992) Plant Cell
4:195-192; Yin et al (1995) Plant J. 7:969-980.
[0154] Plant promoters can be modified, if desired, to affect their
regulatory responsiveness. For example, the CaMV 35S promoter can
be joined to the portion of the RUBISCO gene that represses the
expression of RUBISCO in the absence of light, to create a promoter
that is active in leaves, but not in roots. Constitutive plant
promoters such as actin and ubiquitin, having general expression
properties known in the art, can also be used. See, e.g., McElroy
et al. (1990) Plant Cell 2:163-171; Christensen et al. (1992) Plant
Mol. Biol. 18:675-689.
[0155] Additionally, depending on the desired tissue, expression
can be targeted to the endosperm, aleurone layer, embryo (or its
parts such as scutellum and cotyledons), pericarp, stem, leaves
tubers, roots, etc. Examples of known tissue-specific promoters
include the tuber-directed class I patatin promoter, the promoters
associated with potato tuber ADPGPP genes, the soybean promoter of
beta-conglycinin (7S protein) which drives seed-directed
transcription, and seed-directed promoters from the zein genes of
maize endosperm. See, e.g., Bevan et al. (1986) Nucleic Acids Res.
14:4625-4638; Muller et al. (1990) Mol. Gen. Genet. 224:136-146;
Bray (1987) Planta 172:364-370; and Pedersen et al. (1982) Cell
29:1015-1026. Additional seed-specific promoters include the
phaseolin and napin promoters.
[0156] Recombinant constructs can also include plant-expressible
selectable or screenable marker genes for isolating, identifying or
tracking of plant cells transformed by these constructs. Selectable
markers include, but are not limited to, genes that confer
antibiotic resistances (e.g., resistance to kanamycin or
hygromycin) or herbicide resistance (e.g., resistance to
sulfonylurea, phosphinothricin, or glyphosate). Screenable markers
include, but are not limited to, the genes encoding
beta-glucuronidase (Jefferson (1987) Plant Molec Biol. Rep.
5:387-405), luciferase (Ow et al. (1986) Science 234:856-859), and
the B and C1 gene products that regulate anthocyanin pigment
production (Goff et al. (1990) EMBO J. 9:2517-2522).
[0157] Other elements optionally present in expression vectors
include a replicon that functions in E. coli (or in another
prokaryotic, plant or insect host cell), a selective marker that
functions in a prokaryotic host, e.g., a gene encoding antibiotic
resistance, to permit selection of bacteria that harbor recombinant
plasmids, and unique restriction sites in nonessential regions of
the vector to allow insertion of recombinant sequences.
[0158] Transformation systems for plants as known in the art. See,
e.g., Weissbach & Weissbach, Methods for Plant Molecular
Biology, Academic Press, NY, Section VIII, pp. 421-463 (1988); and
Grierson & Corey, Plant Molecular Biology, 2d Ed., Blackie,
London, Ch. 7-9 (1988). For example, Agrobacterium is often
successfully employed to introduce nucleic acids into plants. Such
transformation preferably uses binary Agrobacterium T-DNA vectors
which can be used to transform dicotyledonous plants,
monocotyledonous plants and plant cells. Bevan (1984) Nuc. Acid
Res. 12:8711-8721; Horsch et al. (1985) Science 227:1229-1231;
Bevan et al. (1982) Ann. Rev. Genet. 16:357-384; Rogers et al.
(1986) Methods Enzymol. 118:627-641; and Hernalsteen et al. (1984)
EMBO J. 3:3039-3041. In embodiments that utilize the Agrobacterium
system for transforming plants, the recombinant DNA constructs
typically comprise at least the right-hand T-DNA border sequence
flanking the DNA sequences to be transformed into the plant cell.
In preferred embodiments, the sequences to be transferred are
flanked by the right- and left-hand T-DNA border sequences. The
design and construction of such T-DNA based transformation vectors
are well known to those skilled in the art.
[0159] Other gene transfer and transformation methods include, but
are not limited to, protoplast transformation through calcium-,
polyethylene glycol (PEG)- or electroporation-mediated uptake of
naked DNA. (see, e.g., Paszlcowski et al. (1984) EMBO J.
3:2717-2722; Potrykus et al. (1985) Molec. Gen. Genet. 199:169-177;
Fromm et al. (1985) Proc. Nat. Acad. Sci. USA 82:5824-5828; and
Shimamoto (1989) Nature 338:274-276); electroporation of plant
tissues (e.g., D'Halluin et al. (1992) Plant Cell 4:1495-1505);
microinjection, silicon carbide-mediated DNA uptake (e.g., Kaeppler
et al. (1990) Plant Cell Reporter 9:415-418), microprojectile
bombardment (e.g., Klein et al. (1988) Proc. Nat. Acad. Sci. USA
85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618);
direct gene transfer, in vitro protoplast transformation, plant
virus-mediated transformation, liposome-mediated transformation,
and ballistic particle acceleration (e.g., Paszkowski et al. (1984)
EMBO J. 3:2717-2722; U.S. Pat. Nos. 4,684,611; 4,407,956;
4,536,475; Crossway et al. (1986) Biotechniques 4:320-334; Riggs et
al. (1986) Proc. Natl. Acad. Sci USA 83:5602-5606; Hinchee et al.
(1988) Biotechnology 6:915-921; and U.S. Pat. No. 4,945,050).
[0160] A wide variety of host cells, plants and plant cell systems
can be used, including, but not limited to, those monocotyledonous
and dicotyledonous plants, such as crops including grain crops
(e.g., wheat, maize, rice, millet, barley), fruit crops (e.g.,
tomato, apple, pear, strawberry, orange), forage crops (e.g.,
alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets,
yam), leafy vegetable crops (e.g., lettuce, spinach); flowering
plants (e.g., petunia, rose, chrysanthemum), conifers and pine
trees (e.g., pine fir, spruce); plants used in phytoremediation
(e.g., heavy metal accumulating plants); oil crops (e.g.,
sunflower, rape seed) and plants used for experimental purposes
(e.g., Arabidopsis).
[0161] Exogenous sequences can also be expressed in seeds (for
example, canola, corn, soybean, rice and barley seed) using
seed-based production techniques, and expression products can be
recovered during seed germination, if desired. See, e.g., PCT
Publication Numbers WO 99/40210; WO 99/16890; WO 99/07206; U.S.
Pat. No. 5,866,121; and U.S. Pat. No. 5,792,933; and all references
cited therein.
[0162] In additional embodiments, fusion molecules (e.g., fusion
proteins) are administered directly to target plant cells (rather
than introducing a nucleic acid encoding a fusion protein). In
certain in vitro situations, target cells are cultured in a medium
containing a fusion molecule as disclosed herein. An important
factor in the administration of polypeptide compounds in plants is
ensuring that the polypeptide has the ability to traverse a cell
wall. However, proteins, viruses, toxins, ballistic methods and the
like have the ability to translocate polypeptides across a plant
cell wall.
[0163] For example, "plasmodesmata" is the term given to explain
cell-to-cell transport of endogenous and viral proteins and
ribonucleoprotein complexes (RNPCs) in plants. Examples of viruses
which can be linked to a fusion molecule for facilitating its
uptake into plant cells include, tobacco mosaic virus (Oparka et
al. (1997) Plant J. 12:781-789); rice phloem thioredoxin
(Ishiwatari et al. (1998) Planta 205:12-22); and potato virus X
(Cruz et al. (1998) Plant Cell 10:495-510). Other suitable chemical
moieties that provide enhanced cellular uptake can also be linked,
either covalently or non-covalently, to fusion molecules to
facilitate penetration of a plant cell. Toxin molecules also have
the ability to transport polypeptides across cell walls.
[0164] Particle-mediated delivery techniques (e.g., ballistic
injection) as described above regarding nucleic acids can also be
used to introduce polypeptides into a plant cell.
Nucleic Acids
[0165] Certain embodiments are directed to nucleic acids. As used
herein, the term nucleic acid refers to both RNA and DNA, including
siRNA, shRNA, miRNA, cDNA, genomic DNA, synthetic (e.g., chemically
synthesized) DNA, as well as naturally occurring and chemically
modified nucleic acids, e.g., synthetic bases or alternative
backbones. A nucleic acid molecule can be double-stranded or
single-stranded (i.e., a sense or an antisense single strand). An
isolated nucleic acid refers to a nucleic acid that is separated
from other nucleic acid bases that are present in a genome,
including nucleic acids that normally flank one or both sides of a
nucleic acid sequence in a vertebrate genome (e.g., nucleic acids
that flank a gene). A conservatively substituted nucleic acid
refers to the substitution of a nucleic acid codon with another
codon that encodes the same amino acid and also refers to nucleic
acids that encode conservatively substituted amino acids, as
described herein with respect to polypeptides. Significantly, the
combination of potential codons for a polypeptide of only about six
residues is manageably small.
[0166] The nucleic acid sequences set forth herein are intended to
represent both DNA and RNA sequences, according to the conventional
practice of allowing the abbreviation "T" stand for "T" or for "U",
as the case may be, for DNA or RNA. Polynucleotides are nucleic
acid molecules of at least three nucleotide subunits.
Polynucleotide analogues or polynucleic acids are chemically
modified polynucleotides or polynucleic acids. In some embodiments,
polynucleotide analogues can be generated by replacing portions of
the sugar-phosphate backbone of a polynucleotide with alternative
functional groups. Morpholino-modified polynucleotides, referred to
herein as "morpholinos," are polynucleotide analogues in which the
bases are linked by a morpholino-phosphorodiamidate backbone (see,
e.g., U.S. Pat. Nos. 5,142,047 and 5,185,444). In addition to
morpholinos, other examples of polynucleotide analogues include
analogues in which the bases are linked by a polyvinyl backbone,
peptide nucleic acids (PNAs) in which the bases are linked by amide
bonds formed by pseudopeptide 2-aminoethyl-glycine groups,
analogues in which the nucleoside subunits are linked by
methylphosphonate groups, analogues in which the phosphate residues
linking nucleoside subunits are replaced by phosphoroamidate
groups, and phosphorothioated DNAs, analogues containing sugar
moieties that have 2' O-methyl group). Polynucleotides of the
invention can be produced through the well-known and routinely used
technique of solid phase synthesis. Alternatively, other suitable
methods for such synthesis can be used (e.g., common molecular
cloning and chemical nucleic acid synthesis techniques). Similar
techniques also can be used to prepare polynucleotide analogues
such as morpholinos or phosphorothioate derivatives. In addition,
polynucleotides and polynucleotide analogues can be obtained
commercially. For oligonucleotides, examples of pharmaceutically
acceptable compositions are salts that include, e.g., (a) salts
formed with cations such as sodium, potassium, ammonium, etc.; (b)
acid addition salts formed with inorganic acids, for example,
hydrochloric acid, hydrobromic acid (c) salts formed with organic
acids e.g., for example, acetic acid, oxalic acid, tartaric acid;
and (d) salts formed from elemental anions e.g., chlorine, bromine,
and iodine.
Kits
[0167] Another aspect of this disclosure relates to kits for
carrying out the administration of a fusion molecule, the
administration of a nucleic acid encoding a fusion polypeptide, the
administration of a nucleoprotein comprising a fusion molecule, or
the administration of a nucleoprotein and a nucleic acid. In one
embodiment, the kit comprises a nucleoprotein comprising a nucleic
acid that is complementary or homologous to a nucleotide sequence
of interest, optionally formulated in a pharmaceutical carrier. In
another embodiment, the kit comprises a nucleoprotein comprising a
nucleic acid that is complementary or homologous to a nucleotide
sequence of interest, and at least one nucleic acid for insertion
into a genome in the vicinity of the sequence of interest,
formulated as appropriate, in one or more separate pharmaceutical
preparations. The nucleoprotein and the nucleic acid can be
formulated together in a single preparation or can be supplied as
separate preparations. Kits may include components and/or
instructions for embodiments as set forth herein, including the
section entitled Additional Description.
[0168] Various publications are cited herein. These publications,
including all patent applications, patents, and journal articles,
are hereby incorporated herein by reference for all purposes; in
the case of conflict, the present specification is controlling.
EXAMPLES
Example 1
Construction of NLS/RecA/Gal4 Fusion Molecule
[0169] A full-length RecA protein, fused at its N-terminus with a
nuclear localization signal (NLS) and at its C-terminus to the Gal4
DNA-binding domain (NLS/RecA/Gal4) was constructed by fusing the
full-length sequence encoding bacterial RecA and the yeast Gal4 DNA
binding domain. Sequences were amplified with the proofreading
polymerase Pfx50 (Invitrogen, Carlsbad, Calif.) from pBEU14 RecA
(Uhlin and Clark, 1981) and pGBT9 Gal4 plasmids (Clontech, Mountain
View, Calif.). The N-terminus of RecA was fused with a nuclear
localization signal (NLS) from SV40 Large T Antigen to promote
nuclear targeting (Keller et al., 2003). NLS in 5' terminal of each
fusion protein was added using the following primer:
5'-CATATGCCACCTAAAAAGAAGAGAAAGGTAGAAGACCCCAAG
ATGGCTATCGACGAAAACAA-3' (SEQ ID NO: 7). The NLS had the amino acid
sequence PPKKKRKVEDPK (SEQ ID NO:1). The Gal4 DNA-binding domain
contained amino acids 1 to 147 of the Gal4 protein and had the
amino acid sequence
MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVES
RLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDM
PLTLRQHRISATSSSEESSNKGQRQLTVS (SEQ ID NO:2). The NLS/RecA/Gal4
fusion protein also contained a His6 tag at carboxy terminus
(521-528 amino acids) to aid in purification. The complete amino
acid sequence of the fusion protein is shown in FIG. 1 (SEQ ID
NO:3). The nucleotide sequence encoding the NLS/RecA/Gal4 fusion
protein is given in FIG. 2 (SEQ ID NO:4).
[0170] The fusion protein was expressed in E. coli
BL21(DE3)pLysS_Cam.sup.+. Protein blot analysis of the lysates
using a goat-anti-RecA antibody showed that the 58 kD fusion
protein was abundantly expressed following induction with 1 mM IPTG
for 2 hours or 16 hours. The protein was purified from lysates of
induced cells using a Ni.sup.+ column.
Example 2
Construction of NLS/RecA
[0171] A NLS/RecA fusion was also constructed by as above for
NLS/RecA/Gal4 except the following primer following 3' primer was
used: 5'-GATCGCGGCCGCAAAATCTTCGTTAGTTTCTG-3', (SEQ ID NO:8). The
amino acid sequence of the NLS/RecA fusion protein is shown in FIG.
3, (SEQ ID NO:5). The nucleotide sequence encoding the NLS/RecA
fusion protein is given in FIG. 4, (SEQ ID NO:6).
Example 3
Formation of Nucleoprotein Filaments with Fusion Molecules
[0172] To test whether the fusion molecules retained the
RecA-mediated ability to form nucleoprotein filaments on
single-stranded DNA, an in vitro reaction, containing the
non-hydrolyzable ATP analogue ATP-.gamma.-S, was used. Stasiak et
al. (1994) Experientia 50:192-203, Baliga et al. (1995) Proc. Natl.
Acad. Sci. USA 92:10393-10397. 10.about.20 ng of dsDNA (.about.250
bp) from the floating head gene in zebrafish diluted in water into
4.0 .mu.l. This DNA was not exposed to Ethidium Bromide (EtBr).
dsDNA was denatured by heating to 95.degree. C. in a temperature
cycler (MASTERCYCLER EPGRADIENT S, Eppendorf, Hamburg, Germany) for
12 minutes and chilled on ice for two minutes. Then was added 0.8
.mu.l of coating buffer (100 mM TrisOAc, pH 7.5; 500 mM NaOAc; 10
mM DTT, 10 mM Mg(OAc).sub.2), 0.6 .mu.l of 16.2 mM ATP.gamma.S
(from Sigma), and 100-200 ng of RecA, NLS-RecA or NLS-RecA-Gal4
into 4.0 .mu.l cssDNA probes. Water was then added to a 7.0 ul
reaction volume that was incubated immediately at 37.degree. C. for
30 minutes to make DNA-RecA filaments.
[0173] The ability of NLS-RecA-Gal4 protein was tested for its
ability to coat single stranded (ss) DNA. An in vitro reaction with
purified protein, single stranded DNA, and a non-hydrolysable form
of ATP, ATP-.gamma.-S was used to test coating activity. For this,
complementary (c), denatured ssDNA (cssDNA) corresponding to the
flh locus was used (250 nucleotides for each strand). Following
coating, the DNA was analyzed on a standard agarose gel for
mobility shift. RecA efficiently coated cssDNA, resulting in a
predicted mobility shift of the single-stranded DNA after
electrophoresis. In contrast, incubation with an equivalent amount
of BSA instead of RecA did not result in a mobility shift of the
DNA (data not shown). Similar to native RecA, the NLS-RecA-Gal4
protein also coated the cssDNA and caused a mobility shift,
indicating that the NLS-RecA-Gal4 fusion protein retained the
ability to bind cssDNA. However, the much of the coated cssDNA
often failed to migrate into the agarose gel, suggesting the
formation of higher order structures between the NLS-RecA-Gal4
filaments. These complexes could be a result of the dimerization
domain in the Gal4 DNA-binding region.
Example 4
Fusion Proteins for Targeted Gene Disruption
[0174] cssDNA RecA filaments produced with different RecA fusion
proteins stimulated targeted gene disruption of chromosomal regions
homologous to the ssDNA
[0175] Design of RecA Filament Experiments.
[0176] Different RecA fusion proteins were tested for the ability
to induce loss of heterozygosity (LOH) at specific loci in
zebrafish. A fusion from the N- to C-termini between the SV40
nuclear localization signal (NLS) from SV40 Large T Antigen, RecA
and the DNA-binding domain of Gal4, NLS-RecA-Gal4 protein (FIG. 5,
panel A), had marked activity to induce LOH at a specific locus in
zebrafish. The NLS sequence was present to enhance nuclear
targeting of ssDNA-RecA filaments. The activity of the
NLS-RecA-Gal4 recombinant protein was compared to NLS-RecA that
lacked the Gal4 domain (FIG. 5, panel B). The Gal4 DNA binding
region contained both a dimerization and metal-binding/DNA
recognition domain that may contribute to the observed activity to
induce LOH at specific loci. For this reason, the activity of
NLS-RecA-Gal4 was compared to an NLS-RecA-Gal4 fusion protein that
lacked the dimerization domain of Gal4 but retains the DNA-binding
domain, termed NLS-RecA-Gal4ADD (FIG. 5, Panel C). cssDNA-RecA
filaments complementary to the golden (gol) locus were injected
into one-cell stage zebrafish embryos that were heterozygous for
the gol.sup.b1 allele (FIG. 6, panel A). Following injection,
embryos were examined for LOH at the gol locus at 3 days post
fertilization (dpf).
[0177] The gol locus was targeted for several reasons. First, the
gol locus is required in a cell-autonomous manner for pigmentation
(Streisinger et al., 1989), providing a direct relationship between
phenotype and genotype and allowing individual cells to be examined
for pigment production. Pigmentation of zebrafish embryos is
visible from day 2 of development (Feitsma et al., 2008). Second,
homozygous recessive gol mutants are viable but lack dark
pigmentation in the retinal epithelial cells and the melanocytes.
gol heterozygotes display normal or wild type levels of
pigmentation (FIG. 6, Panel B). LOH in gol heterozygous embryos
results in clear patches in the eye that lack pigmentation (FIG. 6,
panel B) (Moore et al., 2006). This allows for rapid screening of a
visible phenotype in a mutated gene that is not essential for
embryogenesis. Third, the gene corresponding to the gol mutation,
named slc24a5, is cloned and the genomic region containing the gol
locus is well characterized (Lamason et al., 2005).
Results
[0178] A 1300 bp piece of DNA complementary to a region spanning
from intron 3 to intron 5 of gol gene that contained a mutated exon
4 was used (called gol-1300 in FIG. 6, Panel C). The mutation in
exon 4 results in a premature stop codon and was designed to
replace an endogenous HgaI restriction enzyme recognition site in
exon 4 to create a restriction fragment length polymorphism (RFLP)
if it were recombined into the endogenous gol locus. The 1300 bp
DNA was denatured and coated with NLS-RecA-Gal4 to produce
cssgol-1300-NLS-RecA-Gal4 filaments. Following injection of these
filaments into golb1 heterozygous embryos, 2.9% of the injected
embryos displayed LOH at the gol locus as displayed by loss of
pigmentation in patches of the retinal epithelium (Table I). As a
control, the NLS-RecA-Gal4 protein was mixed with gol-1300 that was
not denatured and injected into golb1 heterozygous embryos. This
condition did not show detectable levels of LOH at the gol locus.
These results indicated that the cssgol-1300-NLS-RecA-Gal4
filaments were able to mutate the wild type copy of gol. We expect
that the frequency at which this occurs is double of what was
detected as disruption of the golb1 allele will not lead to a
detectable phenotype. Analysis of DNA isolated from embryos
containing mosaic patterns of pigmentation did not show a RFLP
(data not shown). This result indicated that the single stranded
DNA in the filament was not substituted into the chromosomal DNA,
i.e., the LOH observed at the gol locus was not induced by
homologous DNA replacement.
[0179] Smaller cssgol-NLS-RecA-Gal4 filaments were also tested and
also found to induce LOH at the gol locus. 60 bp oligos were
designed that were complementary to exon 6, named ssgolex6m-60
sense (s) and ssgolex6m-60 antisense (a) (FIG. 6). Similar to the
gol-1300 fragment, the oligos were designed to contain stop codons
located in the center of the 60 bp sequence and create a RFLP if
the oligos were in replaced by recombination with the endogenous
gol gene. The RFLP would be detected as a change from an endogenous
Afel to artificial HindIII restriction site. When the 60 bp oligos
were coated with NLS-RecA-Gal4 protein, mixed together and injected
into gol heterozygous embryos, LOH was observed in 2.5% of the
injected embryos as missing pigmentation in the eyes, similar to
the frequency observed with the cssgol-1300-NLS-RecA-Gal4
filaments. Again, analysis of DNA isolated from embryos displaying
LOH at the gol locus following injection of
cssgolex6m-60-NLS-RecA-Gal4 filaments did not reveal a RFLP (data
not shown). These results indicated that cssgol-NLS-RecA-Gal4
filaments as short at 60 bp are able to mutate the targeted gene,
but are not induced by recombination.
[0180] To rule out that the induced LOH observed was a result of
recombination of the cssDNA-NLS-RecA-Gal4 filaments, css oligos
without stop codon were tested for the ability to promote LOH at
the gol locus. For this, two adjacent pairs of short css gol
oligos, named golex6-60-1 and -2, were designed complementary to
exon 6 in the gol gene without point mutations (FIG. 6, Panel B).
Injection of either complementary pair of golex6-60-1 or
-2-NLS-RecA-Gal4 filaments into gol heterozygous embryos resulted
in LOH at a frequency similar to the cssgolex6m-60-NLS-RecA-Gal4
filaments (FIG. 6, Panel B and Table I). This frequency was not
enhanced by injection of both cssgolex6-60-1 and -2-NLS-RecA-Gal4
filaments and was dependent upon the NLS-RecA-Gal4 (Table I). These
data support further that the cssDNA-NLS-RecA-Gal4 filaments
promote LOH at the gol locus by a mechanism independent from
recombination resulting in gene replacement.
[0181] A test was made to determine if both complementary
ssDNA-NLS-RecA-Gal4 filaments were required to induce LOH.
Injection of either sense or antisense ssgolex6m-60-NLS-RecA-Gal4
filaments alone into golb1 heterozygous embryos does not induce
detectable LOH at the gol locus (Table I). Furthermore, when
non-paired but adjacent filaments produced with NLS-RecA-Gal4
(either ssgolex6-60-1 sense with ssgolex6-60-2 antisense or
ssgolex6-60-1 antisense with ssgolex6-60-2 sense) were injected
into gol heterozygous embryos, the LOH was not observed (Table I).
These results show that complementary ssDNA NLS-RecA-Gal4 filaments
are required for high frequency induction of LOH at the gol
locus.
[0182] To further test whether 5' phosphate group and 3'OH group
are required for this gene targeting event, two modified pair of
gsg2 oligos were synthesized and injected with NLSRecAGal4. One is
the 5'-amino modified C6 group (5'AmC6) which blocks the phosphate
group on 5' end of oligos. 5' AmC6 was proved no effect for the
binding ability of RecA protein, although it impaired the train
formation of RecA filament (a concatenated form of RecA filament
which can link up to at least 50 oligonucleotides) (Simonson et
al., 1994). The other one is the inverted deoxythymidine 3' end
modifier (3' InvdT) which blocks the 3' hydroxyl group of oligos
and inhibits the DNA polymerase driven primer extension from the 3'
end (Dames et al., 2007). As shown in Table I, injection by either
5'AmC6 or 3'InvdT oligos with NLSRecAGal4, both remain the activity
to induce the LOH event in gol locus under consistent ratio without
compromise, no matter which end is blocked.
[0183] Finally, to confirm the gene targeting specificity caused by
the activity of RecA homologous searching, complementary non-gol
oligos were designed with the same 60nt length for the control
experiment. This pair of non-specific probes was unable to cause
the same LOH phenotype in gol locus as gol oligo-NLS-RecA-Gal4
filaments (Table I). This result shows the requirement of
probe-directed specificity in this NLS-RecA-Gal4 assay.
Example 5
Site-Specific Insertion of an Exogenous Gene
[0184] The above data indicated that the cssDNA-NLS-RecA-Gal4
promoted LOH at the gol locus. Without being bound to a specific
theory, it is believed that this effect was mediated by
double-strand breaks (DSBs) in the targeted region. This example
indicates that the DSB created by NLS-RecA-Gal4 filaments can be
used to promote insertion of supplied exogenous DNA into specific
loci. Without being bound to a specific theory, it is believed that
NLSRecAGal4-filaments cause DSBs in target regions and then
exogenous linear DNA can be incorporated into the site of the break
during repair by the Non-homologous end joining (NHEJ) pathway. In
this example, targeted insertion events were demonstrated by the
tissue-specific expression of EGFP gene after co-injection with
ssDNA-NLS-RecA-Gal4 filaments complementary to the gol, prominin1,
and floating head (flh) loci (FIG. 7). The prominin1 gene was
selected as a previously uncharacterized gene and for its specific
expression in the dorsal diencephalon and retina during embryonic
development. The genes were inserted without promoters, so that
expression requires site-specific site insertion that engages
endogenous promoters that provide for gene expression.
[0185] In order to follow the site-specific targeting event of gene
expression driven by endogenous promoter, the strategy for
targeting is similar to create gene targeting event as the one used
in gol gene disruption as already described. For this, 200 to 300
bp of the target region of the gene is simply amplified by PCR, the
amplification product is denatured by heating, and the single
strands are coated by NLSRecAGal4 with additional co-injection with
a linear fragment of DNA containing an EGFP reporter (FIG. 8). This
type of tissue-specific EGFP expression was observed for 5 to 19%
of the injected embryos from different targeting gene (Table II).
In all cases very little off-target expression was observed (data
not shown).
[0186] For the experiments shown here, the reporter gene is used as
a cassette with a splice acceptor followed by EGFP in all three
frames followed by polyA transcription terminal sequence without
any promoter sequence (FIG. 8, Panel A). Since these reporter genes
could insert in either direction, expression would be expected to
observed in only 1 out of 6 insertions into the targeted gene.
Therefore, it is believed that mutagenic load at the targeted locus
may be at least 6-fold higher than what it can be observed by EGFP
expression after injection. The expression of EGFP in this example
is a marker to indicate the gene disruption event in wild type
fish. Compared with heterozygous fish, homozygous wild type fish
lack LOH phenotype for selection. Consequently, this EGFP insertion
method will allow pre-screening by targeting event to grow to
adulthood. This method is assumed will not only create mutations at
specific loci but also allow following the expression of the
endogenous locus.
[0187] Molecular evidence of targeting and insertion events was
analyzed. Two regions of the gol gene (FIG. 8, Panel A) and one
region of each flh and prominin1 gene were targeted (data not
shown, but similar results were found). As shown below, highly
specific targeted insertions were made into two distinct regions of
the gol gene by choosing two different cssDNA-NLS-RecA-Gal4
filaments. In this example, the location between the two different
probes is about 1 kb (FIG. 8, Panel A). Junction fragments were
amplified between the exogenously supplied EGFP reporter DNA and
the endogenous locus by PCR analysis. This analysis showed
insertion into the gol locus at two distinct sites (FIG. 8, Panel
B). These amplification products were verified by DNA sequencing
and showed the correct sequences from the junction of endogenous
locus and the exogenously supplied DNA. The junctions were found
near the ends of the ssDNA filaments in many cases (FIG. 8, Panel
C). As shown in FIG. 8, Panel C, the site of insertion was within
about 500 bases of the probe. Accordingly, insertion may be made
within about 500 base pairs of an intended site by choice of a
probe
Example 6
Co-Injection of Gol ssDNA-NLS-RecA-Gal4 Filaments with an Exogenous
Gene Results in Mutations that Transmit Through the Germline to the
Next Generation
[0188] Embryos displaying EGFP expression in the eye (FIG. 7) were
selected after co-injection of gol cssDNA-NLS-RecA-Gal4 filaments
with the EGFP reporter gene (FIG. 8) to grow to adulthood.
Gol.sup.b1 homozygous fish were used to make a complementation
testcross with these founder F0 fish. Out of 21 F0 adults screened,
only two F0 fish produced offspring that failed to complement the
b1 allele (FIG. 9). These results demonstrate that this method can
be used to target genes, screen fish, and transmit to germline. The
germline of these two founder fish is highly mosaic, with 0.7 and
3.7% of the offspring from the two founders showing failure to
complement the b1 allele (Table II). One of the F0 founders was
injected with ssDNA-NLS-RecA-Gal4 filaments corresponding to probe
A, and the other was injected with probe B filaments (FIG. 8). The
recovery of two independent insertions at the gol locus using
different probes shows that this method can be used to target any
gene in the genome. Although the fluorescence from the EGFP
reporter gene did not show in non-complementing offspring, the
reason might be consequent with at least a 5-fold less possibility
of in-frame EGFP expression event than gene disruption caused by
DNA deletion or insertion in the gol locus.
Theories of Action
[0189] As per the Examples, it has been demonstrated that
complementary (c) ssDNA-NLS-RecA-Gal4 filaments targeted to the gol
locus are able to induce loss of heterozyogocity at this locus
after injection into zebrafish embryos. This activity requires both
the Gal4 DNA binding/dimerization domains in NLS-RecA-Gal4 and
complementarity between the filaments. Without being bound to a
particular theory, a model is proposed herein for this activity
where the cssDNA-NLS-RecA-Gal4 filaments target the gol locus by
creating arrested replication forks that result in double stranded
breaks (DSBs) (FIG. 10).
[0190] The NLS-RecA-Gal4 protein is able to coat ssDNA (FIG. 8).
When this DNA-protein complex is injected into the zebrafish
embryos, the NLS signal apparently guides the complex into the
nucleus of the embryonic cells. The activity of RecA in the
filaments promotes a homology search to find homologous chromosomal
DNA. Once the chromosomal target is located by homologous pairing,
the cssDNA-NLS-RecA-Gal4 filaments initiate DNA strand invasion.
During the stand invasion and strand exchange steps, the ssDNA
filament will apparently invade and unwind its homologous
double-stranded genomic DNA, resulting in the formation of D-loop
structures. Due to the dimerization domain in Gal4, NLS-RecA-Gal4
is proposed to form a dimer and stabilize the complex of forward
single strand (fss)-NLS-RecA-Gal4 and reverse single strand
(rss)-NLS-RecA-Gal4 filaments on the targeting genomic region (FIG.
10). Because the ssDNA-RecA filament disassembles upon ATP
hydrolysis (Sigurdsson et al., 2002), a non-hydrolytic form of ATP
was used, ATP.gamma.S, for making stable cssDNA-NLS-RecA-Gal4
filaments. It has been noted that some proteins that tightly bind
DNA, such as mutated transcription machinery, can impede
replication fork progression on chromosomes, causing stalled or
collapsed replication forks (Michel et al., 2001; Aguilera and
Gomez-Gonzalez, 2008). Arrested replication forks can increase the
stress on the chromosome, causing the formation of DNA DSB and
replication fork collapse (Michel et al, 1997).
[0191] Arrested replication forks can be processed in
repair-independent and repair-dependent manners. If this
replication fork is not repaired, the free DNA fragment can be
lost, causing a large deletion. Alternatively, the free DNA
fragment may move to a different genomic region, resulting in a
chromosomal translocation (Michel et al., 2001). A large deletion
or translocation can cause a deficient haploid allele during
meiosis in which the resulting phenotype can be detected by a
complementation test or a single-generation haploid screening (Imai
et al., 2000).
[0192] Arrested replication forks can be repaired by a variety of
mechanisms that would be also consistent with our Preliminary
Studies. Stalled replication forks can often trigger cell cycle
arrest and stimulate DSB repair mechanism by either the HR or the
NHEJ pathway. If the arrested replication fork is close to the
telomere, one end of the double strand break (not from the two end
double strand break) created by replication fork collapse can be
repaired by break-induced replication (BIR) pathway (Smith et al.,
2007; Llorente et al., 2008). During this process, the end of the
chromosome is repaired by a form of homologous recombination using
the sister chromosome as a template. This results in a long tract
of LOH.
[0193] Two-end DSBs can also be made upon a replication fork
collapse (Shrivastav et al., 2008). The two-end DSBs result by
resolution of a Holliday junction (HJ) molecule after a "chicken
foot" intermediate structure forms by regression of replication
fork during the initiation of repair (Lundin et al., 2002). It has
been shown that RecA and RecG protein are able to promote DNA
replication fork regression for the DNA repair (Robu et al., 2001
and 2004). Either HR or NHEJ pathway can be used to repair this
kind of two-end DNA DSB (Shrivastav et al., 2008).
[0194] Arrested replication forks are also a target for nuclease
digestion, resulting in the formation of DSB (Michel, et al.,
2001). For example in a Bacteriophage T4 model, T4 Endonuclease VII
cleaves of arrested replication forks induced from an antitumor
drug-topoisomerase complex (Hong and Kreuzer, 2003). In yeast,
Mus81 and Mms4/Eme1 form a heterodimeric structure-specific
endonuclease that can cleave branched DNA structures formed by
Holliday junctions at stalled replication forks (Boddy et al.,
2001; Lundin et al., 2002). This kind of DSB creates a shorter
cleft than ones caused by replication fork collapse. Consequently,
a short DSB is more efficiently paired by either the classical HR
or the NHEJ pathway.
[0195] Therefore, in this model it is proposed that a
cssDNA-NLS-RecA-Gal4 complex or other NLS-recombinase-DNA binding
fusion protein can be used to induce targeted gene disruption by
blockage of the DNA replication fork progression. This results in
site-specific DSB that are repaired by either the endogenous HR or
NHEJ pathway. The homologous searching activity of RecA can provide
target specificity and the Gal4 dimerization domain may stabilize
the complementary joint molecular complex in the targeted region of
chromosome. This DNA-protein complex may also stack with other
cssDNA-NLS-RecA-Gal4 filaments and form a high order structure to
increase the strength of blockage of DNA replication fork. The
arrested replication fork can either cause replication fork
collapse or promote the accessibility of DNA endonucleases to
induce DNA DSBs. This kind of targeted DSB is different from those
randomly induced by stalled replication forks from chemical
inhibition of replication (Feitsma et al., 2008) or mutant DNA
binding proteins (Michel et al., 2001). Based on the results
described herein and the proposed model, it is expected to promote
the site-specific gene mutation by induction of DSB during the
resolution of stalled replication forks. If the DSB is not
repaired, large deletions and translocations are to be expected. If
repaired by the NHEJ pathway, predominantly small deletions or
insertions at the repair site will be observed. If exogenous DNA is
also supplied, it will be inserted into the DSB during its repair
by the NHEJ pathway.
Additional Description
[0196] Embodiments of the invention include a method, kit, use, or
system comprising a fusion protein or proteinaceous fusion molecule
that comprises a polypeptide possessing recombinase activity and a
polypeptide DNA-binding domain. Another embodiment is the system
assembled to be free of any DNA fragment that specifically binds to
the polypeptide DNA-binding domain and/or further comprising an
exogenous DNA that is not specifically bound to the fusion protein.
The embodiments may be further comprising a single-stranded nucleic
acid that forms a nucleoprotein filament by specifically binding to
the polypeptide possessing recombinase activity. The recombinase
may be a recombinase as set forth herein, e.g., RecA, recA803,
uvsX, recA mutants, recA-like recombinases, RuvC, DST2, KEM1 and
XRN1, STPa/DST1, and HPP-1. The polypeptide DNA-binding domain may
be as set forth in this disclosure, e.g., may be chosen from the
group consisting of Gal4, a nuclease, a zinc finger nuclease, a
zinc finger, and a helix-turn-helix protein. The fusion molecule
may further comprise a nuclear localization sequence (NLS). The
exogenous DNA may comprise a DNA marker gene sequence. The
exogenous DNA may encode a polypeptide to be expressed by a cell
that receives the system. The exogenous DNA may be a marker for
identification after insertion into a chromosome of a host cell
that receives the system. The fusion molecule may further comprise
a synthetic linker, optionally disposed between the polypeptide
possessing recombinase activity and the polypeptide DNA-binding
domain, for example a polyethylene oxide. The fusion protein may
comprise RecA and/or Gal4.
[0197] Embodiments include a method, kit, use, or system for
transfection of a target locus of a cell with an exogenous DNA
fragment comprising: a fusion molecule that comprises a polypeptide
possessing recombinase activity and a polypeptide DNA-binding
domain, a single stranded DNA fragment with substantial homology to
the locus (a probe), the fragment specifically binding to the
polypeptide possessing recombinase activity to thereby form a
filament, and an exogenous DNA fragment that is not specifically
bound to the fusion protein, optionally wherein said exogenous DNA
encodes a polypeptide for expression by the cell. The system of may
be free of any DNA fragment that specifically binds to the
polypeptide DNA-binding domain. The system may be provided wherein
the exogenous DNA fragment is present in a molar concentration that
exceeds the molar concentration of the fusion protein, with the
excess optionally being at least 2-fold or between about 2-fold and
500-fold; artisans will immediately appreciate that all ranges and
values between the explicitly stated values are contemplated, e.g.,
10-fold or from about 5-fold to about 50-fold. The exogenous DNA
fragment may encode a polypeptide for cellular expression and/or be
free of a promoter sequence. The exogenous DNA fragment may encode
a polypeptide for cellular expression and optionally include an
expression cassette. The recombinase may be a recombinase as set
forth herein. The polypeptide DNA-binding domain may be chosen from
the group consisting of Gal4, a nuclease, a zinc finger nuclease, a
zinc finger, and a helix-turn-helix protein. The fusion protein may
further comprise a nuclear localization sequence (NLS). The
exogenous DNA may be provided wherein it comprises a DNA marker
gene sequence, encodes a polypeptide to be expressed by a cell that
receives the system, or is a marker for identification after
insertion into a chromosome of a host cell that receives the
system. The fusion protein may further comprise a synthetic linker,
optionally disposed between the polypeptide possessing recombinase
activity and the polypeptide DNA-binding domain, for example a
polyethylene oxide.
[0198] Embodiments include a method, kit, use, or system for
transfecting a cell comprising exposing the cell to the system of
any of 12-21 wherein a user chooses a target site in a chromosome
of the cell, forms the filament, and administers the filament and
the exogenous DNA to the cell, wherein the exogenous DNA is
effectively placed within less than about 5000 basepairs of the
target site; artisans will immediately appreciate that all the
ranges and values within the explicitly stated ranges are
contemplated, e.g., 0-5000, about 100 to about 1000, about 0 to
about 500, less than 2000.
[0199] Embodiments include a method, kit, use, or system for
transfection of a cell with an exogenous and substantially
homologous DNA fragment comprising: a fusion molecule that
comprises a polypeptide possessing recombinase activity and a
polypeptide DNA-binding domain, a double stranded DNA fragment with
at least one portion having a sequence of at least about 20
residues that has substantial homology to the locus, with the
double stranded DNA fragment being free of specific biding to the
fusion protein. The double-stranded DNA fragment may have at least
two sequences of at least 20 residues that have substantial
homology to the locus. The substantial homology may be an identity.
The system may be free of any DNA fragment that specifically binds
to the polypeptide DNA-binding domain. The system may be provided
wherein the double-stranded DNA fragment is present in a molar
concentration that exceeds the molar concentration of the fusion
protein, with the excess optionally being at least 2-fold or
between about 2-fold and 500-fold; artisans will immediately
appreciate that all ranges and values between the explicitly stated
values are contemplated, e.g., 10-fold or from about 5-fold to
about 50-fold. The exogenous DNA fragment may encode a polypeptide
for cellular expression. Thee polypeptide possessing recombinase
may be a recombinase as set forth herein. The polypeptide
DNA-binding domain may be as set forth herein, for example, chosen
from the group consisting of Gal4, a nuclease, a zinc finger
nuclease, a zinc finger, and a helix-turn-helix protein. The fusion
protein may further comprise a nuclear localization sequence (NLS).
The double-stranded DNA may comprises a DNA marker gene sequence,
encode a polypeptide to be expressed by a cell that receives the
system, or a marker for identification after insertion into a
chromosome of a host cell that receives the system. The fusion
protein may further comprise a synthetic linker, optionally
disposed between the polypeptide possessing recombinase activity
and the polypeptide DNA-binding domain, for example a polyethylene
oxide. The homology may comprise homologous sequences located at
the termini of the nucleic acid and/or internally in the first
nucleic acid. Embodiments may include the case wherein the
double-stranded DNA molecule has protruding single-stranded 3'
ends. The polypeptide DNA-binding domain may comprise a Gal4
DNA-binding domain.
[0200] Embodiments include a method, kit, use, or system for
targeted mutagenesis at or near a region of interest in a cellular
nucleic acid sequence, the method comprising: (a) providing a
nucleic acid molecule having homology to the region of interest;
(b) binding a fusion molecule to the nucleic acid, wherein the
fusion molecule comprises: (i) polypeptide sequences having
RecA/Rad51 activity, and (ii) polypeptide sequences comprising a
sequence-specific DNA-binding domain; and (c) introducing the
protein-bound nucleic acid into the cell. The cellular nucleic acid
sequence may be in a chromosome. The nucleic acid molecule may be
DNA. The DNA may be single-stranded. The DNA may be
double-stranded. The nucleic acid molecule may be RNA. The fusion
molecule comprises polypeptide sequences having RecA activity. The
sequence-specific DNA-binding domain may comprise a Gal4
DNA-binding domain. The fusion protein may further comprise a
nuclear localization sequence (NLS). The targeted mutagenesis may
be performed so that it results in conversion of a mutant sequence
to a wild-type sequence. The targeted mutagenesis may be performed
so that it results in conversion of a first allele/haplotype to a
second allele/haplotype. The targeted mutagenesis may be performed
to result in conversion of a wild-type sequence to a mutant
sequence. The mutation may be selected from the group consisting of
a point mutation, an insertion, a deletion, a translocation, and an
inversion.
[0201] Embodiments include a method, kit, use, or system for
targeted homologous recombination between a sequence of interest in
cellular DNA and an exogenous double-stranded nucleic acid, the
method comprising: (a) providing a linear double-stranded nucleic
acid containing one or more regions homologous to the sequence of
interest; (b) binding a fusion protein to the nucleic acid, wherein
the fusion protein comprises: (i) polypeptide sequences having
RecA/Rad51 activity, and (ii) polypeptide sequences comprising a
sequence-specific DNA-binding domain and (c) introducing the
protein-bound nucleic acid into the cell. The nucleic acid of step
(a) may contain the regions homologous to the sequence of interest
at both of its ends. Each of the regions of homology may be at
least 10, 20, or 50 nucleotides in length. One or more regions
homologous to the sequence of interest may be located internally in
the nucleic acid of step (a). Further, the entire exogenous nucleic
acid may be integrated into the cellular DNA. The cellular DNA may
be, for example, in a chromosome, in an episome, comprised of
sequences that encode a protein. The exogenous nucleic acid may
further comprises regulatory sequences. The fusion protein may be
provided so that it comprises polypeptide sequences having RecA
activity. The sequence-specific DNA-binding domain may comprise the
Gal4 DNA-binding domain. The fusion protein may further comprise a
nuclear localization sequence (NLS). The nucleic acid of step (a)
may be provided so that it does not contain a recognition site for
a recombinase, transposase or integrase. The nucleic acid of step
(a) may be free of any transposon or a viral genome. The
recombination may be engineered so that it results in conversion of
a mutant sequence to a wild-type sequence. The recombination may
result in conversion of a first allele/haplotype to a second
allele/haplotype. The recombination may result in conversion of a
wild-type sequence to a mutant sequence. The mutation may be
selected from, for example, the group consisting of a point
mutation, an insertion, a deletion, a translocation and an
inversion.
[0202] An embodiment is a fusion protein comprising: (a)
polypeptide sequences having RecA/Rad51 activity, and (b)
polypeptide sequences comprising a sequence-specific DNA-binding
domain. The fusion protein may further comprise a nuclear
localization sequence (NLS). Alternatively, the fusion molecule may
be placed directly in the cell as described herein.
[0203] Embodiments include a method, kit, use, or system for
stimulating gene conversion in a cell, the method comprising
introducing, into the cell, a fusion molecule as set forth herein.
An example is a proteinaceous fusion molecule comprising: (a) RecA;
(b) a NLS; and (c) Gal4; wherein said gene conversion does not
require hydrolysis of ATP.
[0204] Embodiments include a method, kit, use, or system for
transfecting a cell comprising exposing the cell to an embodiment
of such a system as set forth herein. Embodiments include a method,
kit, use, or system for transfecting a cell comprising exposing the
cell to the system as already described. Examples of cells are a
vertebrate cell, a mammalian cell, a porcine cell, a human cell, a
plant cell and a stem cell. Embodiments include a transgenic animal
formed by the methods or systems described, e.g., a pig or
artiodactyl or mini-pig, goat, rabbit, or mouse. The method, kit,
use, or system may be free of a transposon and/or a viral genome.
The materials may be provided in a pharmaceutically acceptable form
or with a pharmaceutically acceptable excipient. The fusion
molecules may be used to make therapeutic proteins in vitro or in
vivo.
[0205] An embodiment is a method of transfecting a cell comprising
introducing into the cell: an exogenous nucleic acid and a
nucleoprotein filament of a proteinaceous fusion molecule and a
nucleic acid probe complementary to a target site of DNA of the
cell, wherein the fusion protein comprises a recombinase domain
that contributes to the filament, and a DNA-binding domain, wherein
the exogenous nucleic acid is incorporated into the DNA of the cell
and expressed by the cell. An embodiment is a purified composition
for transfection of exogenous DNA into chromosomal DNA of a cell,
the composition comprising a nucleoprotein filament of a probe and
a proteinaceous fusion molecule, wherein the probe comprises
double-stranded denatured DNA complementary to a chromosomal DNA
site, and the fusion molecule comprises a recombinase domain and a
DNA-binding domain, wherein the composition is free of DNA
sequences that specifically bind to the DNA-binding domain. An
embodiment is a method of treating a genetic disease in an animal
comprising introducing into a cell of the animal: an exogenous
nucleic acid and a nucleoprotein filament of a proteinaceous fusion
molecule and a nucleic acid probe complementary to a target site of
DNA of the cell, wherein the fusion molecule comprises a
recombinase domain that contributes to the filament, and a
DNA-binding domain at the time of the introduction, wherein the
exogenous nucleic acid is expressed by the cell to provide a
therapeutic protein to the animal to treat the disease, the method
is performed without a viral vector and without a transposon
vector, and the cell is transfected by a method chosen from the
group consisting of in vitro, in vivo, and ex vivo. The exogenous
nucleic acid may be double-stranded DNA and may also be free of
promoter sequences. The nucleic acid probe may be provided so that
it comprises complementary single strand DNA (cssDNA) and the
DNA-binding domain is not specifically bound to DNA at the time of
the introduction. The exogenous nucleic acid site of insertion may
be provided to be within about 1000 bases of the target site.
Systems are provided with an efficiency of transfecting the cell of
at least about 1%, as measured by an in vitro test with direct
injection. The recombinase may be, or comprise RecA, or a
functional fragment thereof. The DNA-binding domain may comprise
Gal4. The recombinase may be a recombinase set forth herein, e.g.,
chosen from the group consisting of Cre recombinase, Hin
recombinase, Tre recombinase, flippase recombination enzyme, uvsX,
RuvC, DST2, KEM1 and XRN1, STPa/DST1, and HPP-1. The DNA-binding
domain may comprise a polypeptide that specifically binds to DNA
and is chosen from the group consisting of minor groove binders,
major groove binders, antibiotics, intercalating agents,
polyamides, and a polypeptide sequence of a transcription factor,
nuclease, zinc finger nucleases, zinc fingers, and helix-turn-helix
proteins. The fusion molecule may be chosen from the group
consisting of SEQ ID NO:3, SEQ ID NO:5, and conservative
substitutions thereof. The nuclear localization signal may be,
e.g., an SV40 family member. The fusion molecule may comprise a
synthetic non-peptide linker. The probe may be directed to a
mutation in the cell DNA, and the exogenous nucleic acid comprises
a wild-type sequence corresponding to the mutation. The exogenous
nucleic acid may be non-homologous relative to the cell DNA. The
fusion molecule may further comprise a nuclear localization signal
domain. Cells may be transfected with the systems or methods, e.g.,
a vertebrate cell, a mammalian cell, a porcine cell, a human cell,
a plant cell, and a stem cell. A transgenic animal may be formed by
such methods, e.g., from progeny of a germline cell transfected by
the method or systems; e.g., a pig or artiodactyl or mini-pig,
goat, rabbit, or mouse. The method of introduction may be, e.g.,
chosen from the group consisting of electroporation, liposome,
nuclear transplantation, Pronuclear microinjection, and somatic
cell nuclear transfer. The probe may be directed to, for example, a
mutated DNA of the animal that contributes to the disease.
TABLE-US-00001 TABLE I Injection of complementary ssDNA-NLSRecAGal4
results in loss of heterozygocity at the gol (b1) locus RecA type
Probe* Dosage Total Total living Normal dev. Gol eye clones
Injection NLSRecAGal4 css gsg1 45 pg 292 241 N.D 7/241 2.9%.sup.
NLSRecAGal4 ds gsg1 45 pg 284 237 N.D 0/237 0% NLSRecAGal4 ss
gsg2-F 80 pg 155 136 N.D 0/136 0% NLSRecAGal4 ss gsg2-R 80 pg 258
224 N.D 0/224 0% NLSRecAGal4 css gsg2 80 pg 278 201 N.D 5/201
2.5%.sup. No css gbg1 160 pg 74 65 58 0/58 0% No css gbg2 160 pg
227 208 150 0/150 0% No ss gbg1F + gbg2R 160 pg 75 68 68 0/68 0% No
ss gbg2F + gbg1R 160 pg 82 71 70 0/70 0% No css gbg1 + gbg2 160 pg
597 379 342 0/342 0% NLSRecAGal4 css gbg1 160 pg 260 188 144 6/144
4.2%.sup. NLSRecAGal4 css gbg2 160 pg 139 135 107 3/107 2.8%.sup.
NLSRecAGal4 ss gbg1F + gbg2R 160 pg 222 197 190 0/190 0%
NLSRecAGal4 ss gbg2F + gbg1R 160 pg 147 129 129 0/129 0%
NLSRecAGal4 css gbg1 + gbg2 160 pg 979 745 591 20/591 3.4%.sup.
RecA css gbg1 + gbg2 160 pg 317 233 206 0/206 0% NLSRecA css gbg1 +
gbg2 160 pg 301 253 220 0/220 0% NLSRecAGal4.DELTA.DD css gbg1 +
gbg2 160 pg 396 341 296 3/296 1.0%.sup. NLSRecAGal4 css flh oligos
160 pg 298 268 255 0/255 0% NLSRecAGal4 css prim oligos 160 pg 411
314 300 0/300 0% NLSRecAGal4 css vegfa oligos 160 pg 542 337 307
0/307 0% NLSRecAGal4 css 5'AmC6-gbg2 160 pg 152 109 67 2/67
3.0%.sup. NLSRecAGal4 css 3'InvdT-gbg2 160 pg 393 318 236 9/236
3.8%.sup. Non-injection 471 438 N.D 0/438 0% *ss: single strand DNA
probe; ds: double strand DNA probe; css: complementary single
strand DNA probe; gsg: gol probe with stop codon in the middle;
gbg: gol probe without stop codon; 5'AmC6: 5' Amino modifier C6
block; 3'InvdT: 3' Inverted dT block N.D: non-determination
TABLE-US-00002 TABLE II NLSRecAGal4 directed somatic reporter gene
expression into the golden, floating head and prominin-1 Somatic
reporter gene expression Germline complementation test Targeting
Total Founder gol progeny Gene Probe type embryos Surviving
Fluorescence (%) (%) golden gol probe I 791* 649 33 (5.1%) 1/13
(7.7%) 34/919 (3.7%) (gol270bp) gol probe II 207 165 14 (8.5%) 1/8
(12.5%) 1/139 (0.7%) (gol300bp) floating head flh250bp 216 212 18
(8.5%) prominin-1 prim200bp 162 154 29 (18.8%) *Data were collected
from three individual experiments
Sequence CWU 1
1
8112PRTUnknownmammalian 1Pro Pro Lys Lys Lys Arg Lys Val Glu Asp
Pro Lys 1 5 10 2147PRTUnknownmammalian 2Met Lys Leu Leu Ser Ser Ile
Glu Gln Ala Cys Asp Ile Cys Arg Leu 1 5 10 15 Lys Lys Leu Lys Cys
Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu 20 25 30 Lys Asn Asn
Trp Glu Cys Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro 35 40 45 Leu
Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu 50 55
60 Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile
65 70 75 80 Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr
Gly Leu 85 90 95 Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr
Asp Arg Leu Ala 100 105 110 Ser Val Glu Thr Asp Met Pro Leu Thr Leu
Arg Gln His Arg Ile Ser 115 120 125 Ala Thr Ser Ser Ser Glu Glu Ser
Ser Asn Lys Gly Gln Arg Gln Leu 130 135 140 Thr Val Ser 145
3528PRTartificialSynthesized 3Met Pro Pro Lys Lys Lys Arg Lys Val
Glu Asp Pro Lys Met Ala Ile 1 5 10 15 Asp Glu Asn Lys Gln Lys Ala
Leu Ala Ala Ala Leu Gly Gln Ile Glu 20 25 30 Lys Gln Phe Gly Lys
Gly Ser Ile Met Arg Leu Gly Glu Asp Arg Ser 35 40 45 Met Asp Val
Glu Thr Ile Ser Thr Gly Ser Leu Ser Leu Asp Ile Ala 50 55 60 Leu
Gly Ala Gly Gly Leu Pro Met Gly Arg Ile Val Glu Ile Tyr Gly 65 70
75 80 Pro Glu Ser Ser Gly Lys Thr Thr Leu Thr Leu Gln Val Ile Ala
Ala 85 90 95 Ala Gln Arg Glu Gly Lys Thr Cys Ala Phe Ile Asp Ala
Glu His Ala 100 105 110 Leu Asp Pro Ile Tyr Ala Arg Lys Leu Gly Val
Asp Ile Asp Asn Leu 115 120 125 Leu Cys Ser Gln Pro Asp Thr Gly Glu
Gln Ala Leu Glu Ile Cys Asp 130 135 140 Ala Leu Ala Arg Ser Gly Ala
Val Asp Val Ile Val Val Asp Ser Val 145 150 155 160 Ala Ala Leu Thr
Pro Lys Ala Glu Ile Glu Gly Glu Ile Gly Asp Ser 165 170 175 His Met
Gly Leu Ala Ala Arg Met Met Ser Gln Ala Met Arg Lys Leu 180 185 190
Ala Gly Asn Leu Lys Gln Ser Asn Thr Leu Leu Ile Phe Ile Asn Gln 195
200 205 Ile Arg Met Lys Ile Gly Val Met Phe Gly Asn Pro Glu Thr Thr
Thr 210 215 220 Gly Gly Asn Ala Leu Lys Phe Tyr Ala Ser Val Arg Leu
Asp Ile Arg 225 230 235 240 Arg Ile Gly Ala Val Lys Glu Gly Glu Asn
Val Val Gly Ser Glu Thr 245 250 255 Arg Val Lys Val Val Lys Asn Lys
Ile Ala Ala Pro Phe Lys Gln Ala 260 265 270 Glu Phe Gln Ile Leu Tyr
Gly Glu Gly Ile Asn Phe Tyr Gly Glu Leu 275 280 285 Val Asp Leu Gly
Val Lys Glu Lys Leu Ile Glu Lys Ala Gly Ala Trp 290 295 300 Tyr Ser
Tyr Lys Gly Glu Lys Ile Gly Gln Gly Lys Ala Asn Ala Thr 305 310 315
320 Ala Trp Leu Lys Asp Asn Pro Glu Thr Ala Lys Glu Ile Glu Lys Lys
325 330 335 Val Arg Glu Leu Leu Leu Ser Asn Pro Asn Ser Thr Pro Asp
Phe Ser 340 345 350 Val Asp Asp Ser Glu Gly Val Ala Glu Thr Asn Glu
Asp Phe Ala Ser 355 360 365 Met Lys Leu Leu Ser Ser Ile Glu Gln Ala
Cys Asp Ile Cys Arg Leu 370 375 380 Lys Lys Leu Lys Cys Ser Lys Glu
Lys Pro Lys Cys Ala Lys Cys Leu 385 390 395 400 Lys Asn Asn Trp Glu
Cys Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro 405 410 415 Leu Thr Arg
Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu 420 425 430 Glu
Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile 435 440
445 Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu
450 455 460 Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg
Leu Ala 465 470 475 480 Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg
Gln His Arg Ile Ser 485 490 495 Ala Thr Ser Ser Ser Glu Glu Ser Ser
Asn Lys Gly Gln Arg Gln Leu 500 505 510 Thr Val Ser Ala Ala Ala Leu
Glu His His His His His His His His 515 520 525
41584DNAArtificialsynthesized 4atgccaccta aaaagaagag aaaggtagaa
gaccccaaga tggctatcga cgaaaacaaa 60cagaaagcgt tggcggcagc actgggccag
attgagaaac aatttggtaa aggctccatc 120atgcgcctgg gtgaagaccg
ttccatggat gtggaaacca tctctaccgg ttcgctttca 180ctggatatcg
cgcttggggc aggtggtctg ccgatgggcc gtatcgtcga aatctacgga
240ccggaatctt ccggtaaaac cacgctgacg ctgcaggtga tcgccgcagc
gcagcgtgaa 300ggtaaaacct gtgcgtttat cgatgctgaa cacgcgctgg
acccaatcta cgcacgtaaa 360ctgggcgtcg atatcgataa cctgctgtgc
tcccagccgg acaccggcga gcaggcactg 420gaaatctgtg acgccctggc
gcgttctggc gcagtagacg ttatcgtcgt tgactccgtg 480gcggcactga
cgccgaaagc ggaaatcgaa ggcgaaatcg gcgactctca catgggcctt
540gcggcacgta tgatgagcca ggcgatgcgt aagctggcgg gtaacctgaa
gcagtccaac 600acgctgctga tcttcatcaa ccagatccgt atgaaaattg
gtgtgatgtt cggtaacccg 660gaaaccacta ccggtggtaa cgcgctgaaa
ttctacgcct ctgttcgtct cgacatccgt 720cgtatcggcg cggtgaaaga
gggcgaaaac gtggtgggta gcgaaacccg cgtgaaagtg 780gtgaagaaca
aaatcgctgc gccgtttaaa caggctgaat tccagatcct ctacggcgaa
840ggtatcaact tctacggcga actggttgac ctgggcgtaa aagagaagct
gatcgagaaa 900gcaggcgcgt ggtacagcta caaaggtgag aagatcggtc
agggtaaagc gaatgcgact 960gcctggctga aagataaccc ggaaaccgcg
aaagagatcg agaagaaagt acgtgagttg 1020ctgctgagca acccgaactc
aacgccggat ttctctgtag atgatagcga aggcgtagca 1080gaaactaacg
aagattttgc tagcatgaag ctactgtctt ctatcgaaca agcatgcgat
1140atttgccgac ttaaaaagct caagtgctcc aaagaaaaac cgaagtgcgc
caagtgtctg 1200aagaacaact gggagtgtcg ctactctccc aaaaccaaaa
ggtctccgct gactagggca 1260catctgacag aagtggaatc aaggctagaa
agactggaac agctatttct actgattttt 1320cctcgagaag accttgacat
gattttgaaa atggattctt tacaggatat aaaagcattg 1380ttaacaggat
tatttgtaca agataatgtg aataaagatg ccgtcacaga tagattggct
1440tcagtggaga ctgatatgcc tctaacattg agacagcata gaataagtgc
gacatcatca 1500tcggaagaga gtagtaacaa aggtcaaaga cagttgactg
tatcggcggc cgcactcgag 1560caccaccacc accaccacca ccac
15845381PRTArtificialsynthesized 5Met Pro Pro Lys Lys Lys Arg Lys
Val Glu Asp Pro Lys Met Ala Ile 1 5 10 15 Asp Glu Asn Lys Gln Lys
Ala Leu Ala Ala Ala Leu Gly Gln Ile Glu 20 25 30 Lys Gln Phe Gly
Lys Gly Ser Ile Met Arg Leu Gly Glu Asp Arg Ser 35 40 45 Met Asp
Val Glu Thr Ile Ser Thr Gly Ser Leu Ser Leu Asp Ile Ala 50 55 60
Leu Gly Ala Gly Gly Leu Pro Met Gly Arg Ile Val Glu Ile Tyr Gly 65
70 75 80 Pro Glu Ser Ser Gly Lys Thr Thr Leu Thr Leu Gln Val Ile
Ala Ala 85 90 95 Ala Gln Arg Glu Gly Lys Thr Cys Ala Phe Ile Asp
Ala Glu His Ala 100 105 110 Leu Asp Pro Ile Tyr Ala Arg Lys Leu Gly
Val Asp Ile Asp Asn Leu 115 120 125 Leu Cys Ser Gln Pro Asp Thr Gly
Glu Gln Ala Leu Glu Ile Cys Asp 130 135 140 Ala Leu Ala Arg Ser Gly
Ala Val Asp Val Ile Val Val Asp Ser Val 145 150 155 160 Ala Ala Leu
Thr Pro Lys Ala Glu Ile Glu Gly Glu Ile Gly Asp Ser 165 170 175 His
Met Gly Leu Ala Ala Arg Met Met Ser Gln Ala Met Arg Lys Leu 180 185
190 Ala Gly Asn Leu Lys Gln Ser Asn Thr Leu Leu Ile Phe Ile Asn Gln
195 200 205 Ile Arg Met Lys Ile Gly Val Met Phe Gly Asn Pro Glu Thr
Thr Thr 210 215 220 Gly Gly Asn Ala Leu Lys Phe Tyr Ala Ser Val Arg
Leu Asp Ile Arg 225 230 235 240 Arg Ile Gly Ala Val Lys Glu Gly Glu
Asn Val Val Gly Ser Glu Thr 245 250 255 Arg Val Lys Val Val Lys Asn
Lys Ile Ala Ala Pro Phe Lys Gln Ala 260 265 270 Glu Phe Gln Ile Leu
Tyr Gly Glu Gly Ile Asn Phe Tyr Gly Glu Leu 275 280 285 Val Asp Leu
Gly Val Lys Glu Lys Leu Ile Glu Lys Ala Gly Ala Trp 290 295 300 Tyr
Ser Tyr Lys Gly Glu Lys Ile Gly Gln Gly Lys Ala Asn Ala Thr 305 310
315 320 Ala Trp Leu Lys Asp Asn Pro Glu Thr Ala Lys Glu Ile Glu Lys
Lys 325 330 335 Val Arg Glu Leu Leu Leu Ser Asn Pro Asn Ser Thr Pro
Asp Phe Ser 340 345 350 Val Asp Asp Ser Glu Gly Val Ala Glu Thr Asn
Glu Asp Phe Ala Ser 355 360 365 Ala Ala Ala Leu Glu His His His His
His His His His 370 375 380 61137DNAArtificialsynthesized
6atgccaccta aaaagaagag aaaggtagaa gaccccaaga tggctatcga cgaaaacaaa
60cagaaagcgt tggcggcagc actgggccag attgagaaac aatttggtaa aggctccatc
120atgcgcctgg gtgaagaccg ttccatggat gtggaaacca tctctaccgg
ttcgctttca 180ctggatatcg cgcttggggc aggtggtctg ccgatgggcc
gtatcgtcga aatctacgga 240ccggaatctt ccggtaaaac cacgctgacg
ctgcaggtga tcgccgcagc gcagcgtgaa 300ggtaaaacct gtgcgtttat
cgatgctgaa cacgcgctgg acccaatcta cgcacgtaaa 360ctgggcgtcg
atatcgataa cctgctgtgc tcccagccgg acaccggcga gcaggcactg
420gaaatctgtg acgccctggc gcgttctggc gcagtagacg ttatcgtcgt
tgactccgtg 480gcggcactga cgccgaaagc ggaaatcgaa ggcgaaatcg
gcgactctca catgggcctt 540gcggcacgta tgatgagcca ggcgatgcgt
aagctggcgg gtaacctgaa gcagtccaac 600acgctgctga tcttcatcaa
ccagatccgt atgaaaattg gtgtgatgtt cggtaacccg 660gaaaccacta
ccggtggtaa cgcgctgaaa ttctacgcct ctgttcgtct cgacatccgt
720cgtatcggcg cggtgaaaga gggcgaaaac gtggtgggta gcgaaacccg
cgtgaaagtg 780gtgaagaaca aaatcgctgc gccgtttaaa caggctgaat
tccagatcct ctacggcgaa 840ggtatcaact tctacggcga actggttgac
ctgggcgtaa aagagaagct gatcgagaaa 900gcaggcgcgt ggtacagcta
caaaggtgag aagatcggtc agggtaaagc gaatgcgact 960gcctggctga
aagataaccc ggaaaccgcg aaagagatcg agaagaaagt acgtgagttg
1020ctgctgagca acccgaactc aacgccggat ttctctgtag atgatagcga
aggcgtagca 1080gaaactaacg aagattttgc ggccgcactc gagcaccacc
accaccacca ccaccac 1137762DNAArtificialprimer 7catatgccac
ctaaaaagaa gagaaaggta gaagacccca agatggctat cgacgaaaac 60aa
62832DNAArtificialprimer 8gatcgcggcc gcaaaatctt cgttagtttc tg
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