FIELD OF THE INVENTION

This invention relates to the field of gene targeting in vertebrate cells by homologous recombination using gene targeting vectors, including vectors based on adeno-associated virus (AAV).

BACKGROUND TO THE INVENTION

Early methods for introducing defined mutations into mammalian chromosomes by gene targeting involve the transfection, electroporation or microinjection (Smithies et al. (1985) Nature 317: 230-234; Thomas et al. (1986) Cell 44: 419-428) of plasmid DNA. These plasmid- based methods, except for microinjection, produce homologous recombination events in only a small fraction of the total cell population; Thomas and Capecchi (1987) Cell 51: 503- 512). Plasmid-based homologous recombination techniques have been used primarily to create transgenic mice from embryonic stems cells. However, in stem cells, recombination frequencies are naturally very high to support DNA repair during cell-division and thus they are amenable to gene targeting. In non-dividing or differentiated somatic cell types, this process is essentially shut off, making these cells more difficult to target.

More recently, WO 98/48005 disclosed the use of parvoviral vectors, particularly Adeno- Associated Virus (AAV) vectors, for homologous recombination. These vectors are far more efficient at gene-targeting in human somatic cell-types than the older DNA plasmid-based vectors. Thus AAV vectors are now routinely used for gene targeting applications and have allowed the routine creation of modified somatic cell lines with a precisely modified target gene without off-target genetic changes. These cell lines are the most accurate in-vitro models available to researchers to date, and are facilitating the development of new personalized drugs and diagnostics, particularly in the field of cancer.

Although the discovery and use of AAV as a gene targeting vector provided a marked improvement over the early methods of gene targeting, particularly for somatic cells, the efficiency of this process is still relatively low, only approximately 1% of somatic cells that are exposed to the targeting vector are modified at the correct location in the genome. This means that significant time, skill and resource are needed to generate a single successfully targeted cell line.

Thus, a need exists for gene targeting vectors and methods for obtaining a specific genetic modification at selected target sites in vertebrate cellular genomes at higher efficiency.

Typically, a gene targeting vector such as a plasmid or gene targeting viral vector, such as AAV, will utilize a targeting construct that includes the genetic modification to be introduced and a positive selection cassette flanked by regions of homology (genomic DNA sequence). One of the problems associated with existing gene targeting methods, particularly in somatic cells, is the need to identify cells that are correctly targeted against a background of cells that are either not transduced by the vector, contain vector present episomally, or that have incorporated the targeting construct into a random position in the genome, so called "off-targets". Typically, identifying correctly targeted cells involves selecting for cells based on the expression of a positive selection cassette, e.g. neo, and then further analysing each of the selected cells, or clones, to determine whether these have incorporated the introduced DNA sequence at the correct position in the genome. This can be a lengthy process which usually involves PCR and/or Southern blots.

One strategy that has been used to improve the efficiency of plasmid gene targeting vectors, such as those used to modify mouse embryonic stem cells, includes the usual positive selection cassette (e.g. neo) flanked by genomic DNA but also has a thymidine kinase gene (TK) expression cassette (negative selection cassette) at the distal end of the genomic DNA. This permits selection against the presence of TK in the genome (using gancyclovir), in addition to selection for G418 (neo) resistance. Clones positive for TK cannot have undergone a correct homologous recombination event and are negatively selected. Ideally, survivors of gancyclovir selection are thus enriched for homologous recombinants. However, such a strategy is only applicable to plasmid gene targeting vectors as these have sufficiently large genome sizes to permit the incorporation of both expression cassettes and the regions of homology necessary to effect homologous recombination. It also requires the application of a secondary selection agent (gancyclovir).

RNAi expression cassettes have been incorporated into rAAV gene transfer vectors, see Grimm et al., Methods in Enzymology 02/2005; 392:381-405. However, there have been no reports of the successful use of gene targeting vectors that include RNAi cassettes, or that utilise these to enhance the efficiency of gene targeting.

An abstract, A. M. Cornea, D. W. Russell, Journal of Investigative Medicine: 1 January 2005 - Volume 53 - Issue 1 - p S84, WESTERN ABSTRACTS: Western Student Medical Research Forum Student Scientific Session IV 1:30 PM: Thursday, February 3, 2005, suggested that, in theory, the frequency of random integrations of AAV gene targeting vectors could be reduced by adding a negative selection marker in the form of an RNAi cassette targeted to either the CDK2 or LMNB1 gene of the host cell. However, the abstract does not provide sufficient detail of the vector design to enable the skilled man to construct the AAV vectors, or report any data showing that the proposed AAV gene targeting vectors were actually made and successfully used to reduce the frequency of random integrations. Thus there remains a need in the art for new gene targeting vectors and methods to permit the generation of precisely modified cells at higher efficiency. SUMMARY OF THE INVENTION

The present invention provides a gene targeting vector for introducing a genetic modification at a preselected genomic target locus by homologous recombination in a vertebrate host cell comprising: a. a targeting construct comprising the genetic modification flanked by regions of homology which are substantially identical to the genomic target locus, said regions each being at least 50bp in length;

b. at least one RNAi expression cassette positioned outside the region of

homology.

The RNAi expression cassette may be positioned between the vector end, or ITR, and the region of homology. The gene targeting vector may be a nucleic acid construct, including a PCR product, a plasmid, or a viral gene targeting vector such as a retroviral, adenoviral or parvoviral vector.

The gene targeting vector may be a parvoviral gene targeting vector, such as AAV (adeno associated virus) that includes at least one parvoviral inverted terminal repeat (ITR) flanking the targeting construct, the RNAi expression cassette being positioned between the region of homology and the ITR.

The RNAi expression cassette may be a negative selection cassette such that cells that have randomly integrated the RNAi expression cassette are negatively selected. Suitable RNAi negative selection cassettes include those that express an RNAi that targets a host cell gene that is essential for cell survival and/or proliferation, the host cell gene may be an endogenous host cell gene, for example, a housekeeping gene, a cell cycle gene, or an anti- apoptotic gene. Suitable genes include CDK2, PLK1, PMSA1, or RPL3. The RNAi may target a host cell gene the down regulation of which confers sensitivity to a selection agent. This means that a cell that has randomly integrated the RNAi expression cassette (as an off-target), or that includes vector(s) present episomally, is sensitised to a selection agent and may be negatively selected by exposure to the selection agent. The host cell gene may be an endogenous gene such as HPRT, Thymidine Kinase, APRT or XGPRT. The host cell gene may be a heterologous gene, for example, that was previously introduced into the host cell to confer sensitivity to a selection agent.

The RNAi may target a host cell gene (endogenous or heterologous) that confers a selectable phenotype, for example, a cell surface marker the presence or absence of which permits selection of cells that include the RNAi expression cassette (off-targets).

The gene targeting vectors can also include a positive selection marker as part of the gene targeting construct. The positive selection marker may be located in the conventional location, towards the centre of the targeting construct so that it is flanked on both sides by the regions of homology. The vectors may comprise an RNAi that targets the positive selection marker. The RNAi expression cassette may thus target the positive selection marker such that cells that have randomly integrated the vector, or that include vectors present episomally, are negatively selected due to suppression of expression of the positive selection marker.

The RNAi expression cassette may target a host cell gene that normally functions to inhibit homologous recombination, such that homologous recombination is increased by expression of the RNAi, for example, the host cell gene may be in the cNHEJ, aNHEJ, ssDNA binding protein, homologous recombination or mismatch repair pathways. Suitable genes include DNAPKcs, MLH1, MSH2, BLM, PCNA, RPA, POLD1, POLD2, POLD3, and POLD4.

The vector may comprise multiple RNAi expression cassettes, for example, at least one RNAi expression cassette flanking either side of the homologous region. This may be multiple RNAi cassettes directed to the same target, or RNAi cassettes directed to different targets. For example, the vector may include an RNAi negative selection cassette and an RNAi that targets a host cell gene that normally functions to inhibit homologous recombination, such as a gene in the MMR pathway.

The vector may comprise an RNAi expression cassette that is selected from shRNA, siRNA or miRNA.

It is believed that the inclusion of the RNAi expression cassette should increase the efficiency of the process sufficiently to enable the use of gene targeting vectors that do not require a positive selection cassette. Thus the vectors of the invention may be "scarless" such that the targeting construct does not include a positive selection marker, for example, the targeting construct consists essentially of the genetic modification to be introduced flanked by regions of homology which are substantially identical to the genomic target locus, said regions each being at least 50 bp in length. The gene targeting vectors of the present invention may be in the form of unpackaged vector genomes or may be packaged as virus. Thus the present invention also provides recombinant viruses, particularly recombinant parvoviruses comprising any of the vectors according to the invention. The recombinant viruses may comprise a chimeric or modified capsid.

The present invention also provides methods of the using any of the gene targeting vectors according to the invention and includes a method of producing a vertebrate cell having a genetic modification at a preselected genomic target locus, the method comprising: a) introducing into the vertebrate cell an effective amount of a gene targeting

vector comprising, i) a targeting construct comprising the genetic modification

flanked by regions of homology which are substantially identical to the genomic target locus, said regions each being at least 50bp in length; ii) at least one RNAi expression cassette positioned outside

the region of homology;

iii) optionally, a positive selection marker; and b. determining if the genomic target locus has the introduced genetic modification.

The present invention also provides methods of using any of the parvovirus gene targeting vectors according to the invention and includes a method of producing a vertebrate cell having a genetic modification at a preselected genomic target locus, the method comprising: a) introducing into the vertebrate cell an effective amount of a recombinant

parvovirus comprising, i) a targeting construct comprising the genetic modification

flanked by regions of homology which are substantially identical to the genomic target locus, said regions each being at least 50bp in length;

at least one parvoviral inverted terminal repeat (ITR) flanking the targeting construct;

ii) at least one RNAi expression cassette positioned

between the region of homology and the ITR;

v) optionally, a positive selection marker; and b) determining if the genomic target locus has the introduced genetic

modification.

The methods of the invention may also include the step of selecting for cells that have not incorporated the RNAi expression cassette, for example, by exposing the cell to a selection agent to negatively select cells that comprise the RNAi expression cassette.

The methods of the invention may further comprise the step of selecting for cells that have introduced the positive selection marker, for example, by exposing the cells to a selection agent.

The methods of the present invention may further comprise the step of removing the positive selection marker.

The methods of the present invention may further comprise the step of producing a cell line from a cell that has the introduced genetic modification.

The methods of the present invention include methods wherein the targeting frequency is at least 3%.

The vectors and methods of the invention may be particularly advantageous when applied to somatic cell lines. The present invention also includes vertebrate cell lines comprising a genetic modification at a preselected genomic target locus which has been modified by the vectors or methods of the present invention. These cell lines may be "scarless" cell lines as they comprise the introduced genetic modification but no other modifications are introduced, for example, no markers or other non-homologous sequences.

The present invention also includes cell line pairs comprising a genetically modified cell line modified by the vectors and/or methods of the present invention and the parental cell line that was used to generate the genetically modified cell line.

The present invention also includes gene targeting kits comprising the vectors according to present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Schematic depiction of the targeting vectors used in the example experiments to introduce the D145N point mutation into the CDK2 gene.

Figure 2: Bar chart showing that HPRT shRNA negative selection cassettes increase the number of correctly targeted cells in rAAV-mediated gene targeting..

Figure 3: Schematic diagram showing variant designs of rAAV shRNA negative selection vectors.

Figure 4: Figure 4A -Schematic diagram showing a rAAV gene targeting vector with a shRNA targeting the positive selection cassette on the vector. Figure 4B - bar chart showing that the use of this vector decreases the recovery of random integrants.

Figure 5: Figures 5A and 5B - bar charts showing a rAAV gene targeting vector with dual HPRT negative selection cassettes in combination with MMR pathway inhibition (MSH2 inhibition) increases gene targeting frequency in two MMR proficient cell lines. Figure 5C - western blot showing inhibition of MSH2. Figure 5D -schematic diagram of rAAV gene targeting vector with dual HPRT shRNA negative selection cassettes..

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides gene targeting vectors to introduce a genetic modification at a preselected genomic target locus by homologous recombination in a vertebrate host cell comprising: a. a targeting construct comprising the genetic modification flanked by regions of homology which are substantially identical to the genomic target locus, said regions each being at least 50bp in length; and

b. at least one RNAi expression cassette positioned outside the region of homology. The RNAi expression cassette is thus positioned so that it may be introduced into the host cell genome by a random integration event and not by homologous recombination. Typically it is located between the vector end, or ITR, and the region of homology.

The gene targeting vector may be a nucleic acid construct to be used to introduce a genetic modification into the host cell, including a PCR product, a plasmid, or viral vector such as a retroviral, adenoviral, parvoviral or other viral vector.

Parvoviral gene targeting vectors, including AAV and those disclosed in WO 98/40085, may be particularly useful in the practice of the invention. These vectors include at least one parvoviral inverted terminal repeat (ITR) flanking the targeting construct, or more typically, an ITR sequence at each end of the genome, with the RNAi expression cassette being positioned between the region of homology and the ITR.

The regions of homology are substantially identical to the genomic target locus and comprise a genomic DNA sequence that is homologous to the host cell genome. The provision of such sequences is well understood in the field of gene targeting. The use of PCR is a common method for generating genomic sequence from a genomic target locus and desired mutations can be introduced by use of alterations located in the PCR primer sequence. Alternatively, a PCR product derived from a genomic target locus can be cloned into a plasmid and mutations introduced through use of a commercially available site directed mutagenesis kit.

As is understood by the skilled man, the regions of homology may vary in length, for example, depending on the capacity of the vector used or the size of the modification to be introduced. However, each region of homology may be greater than 50bp, or greater than lOObp, or greater than 200bp. Upon entry of the vector into the cell, homologous pairing occurs between the targeting construct and the target locus, resulting in the modifications being introduced into the target locus. The modification can include one or more deletions, insertions, substitutions, or a combination thereof.

The RNAi expression cassette is positioned outside the regions of homology such that it may be introduced into the host cell genome by a random integration event and not by homologous recombination. Thus correctly targeted cells will not incorporate the RNAi expression cassette into their genome whilst incorrectly targeted cell will incorporate the RNAi. The RNAi expression cassette may also remain in the host cell on gene targeting vectors that are present episomally.

The RNAi expression cassette may be a negative selection cassette so that cells that have randomly integrated the RNAi expression cassette, or have the RNAi present episomally are negatively selected. The expression of RNAi from such a cassette may kill or inhibit the proliferation of cells that include the cassette. This means that the surviving cell population is enriched with cells that do not express the RNAi and should include correctly targeted cells. The negative selection may occur as a direct result of the RNAi expression or may require the addition of a selection agent.

Examples of RNAi negative selection cassettes include RNAi that targets a host cell gene that is essential for cell survival and/or proliferation, the host cell gene may be an endogenous host cell gene, for example, a housekeeping gene, a cell cycle gene, or an anti-apoptotic gene, for example, the gene may be CDK2, PLK1, PSMA1, or RPL3.

However, in some embodiments the vectors and methods of the invention do not include AAV gene targeting vectors that comprise at least one RNAi expression cassette targeted to CDK2 or LMNBl. Such a vector may incorporate any of the other features described herein and be used in any of the methods described herein.

I n some embodiments the vectors of the invention comprise a gene targeting vector for introducing a genetic modification at a preselected genomic target locus by homologous recombination in a vertebrate host cell comprising: a. a targeting construct comprising the genetic modification flanked by regions of homology which are substantially identical to the genomic target locus, said regions each being at least 50bp in length;

b. at least one RNAi expression cassette positioned outside the

the region of homology ; and wherein the gene targeting vector does not comprise at least one RNAi expression cassette targeted to the endogenous host cell genes CDK2 or LMNBl. Such a vector may incorporate any of the other features described herein and be used in any of the methods described herein.

The RNAi may target a host cell gene such that a cell that has randomly integrated the RNAi expression cassette and/or that includes vector(s) present episomally, is sensitised to a selection agent. The host cell gene may be an endogenous host cell gene such as HPRT, Thymidine Kinase, APRT or XGPRT. The RNAi produced by the intact expression cassette on the vector can reduce the level of, for example, endogenous HPRT, thus sensitising the cell to negative selection using HAT media. Alternatively, the host cell gene may be a heterologous gene that was previously introduced into the cell to confer resistance to a selection agent. Examples of such genes include hygromycin, puromycin, and neomycin. Vectors and methods using an RNAi expression cassette that targets a heterologous gene in this way may be particularly useful for engineering bioproducer cell lines.

The RNAi may target a host cell gene (endogenous or heterologous) that confers a selectable phenotype, for example, a cell surface marker that presence or absence of which permits the identification of cells that include the RNAi expression cassette (off-targets). Suitable markers include those that allow visual detection and elimination of off-targets from the cell population and/or permit automated cell sorting, such as fluorescent proteins. Examples of such genes include any of the CD antigen family of proteins which are expressed on the surface of the cell and for which fluorescently labelled antibodies can be used to indicate the surface expression (or lack thereof) for use in FACS sorting.

The vectors and methods of the invention may also include a positive selection marker. The positive selection marker may be any suitable positive selection marker and may be positioned, in the conventional manner, towards the centre of the gene targeting construct such that is incorporated into the host genome by an on-target homologous recombination event. Particularly useful markers include hygromycin, puromycin and neomycin. The vectors and methods of the invention may utilise an RNAi targeted to an endogenous or heterologous gene to provide negative selection of incorrectly targeted cells and a conventional positive selection marker to identify surviving cells that are correctly targeted. There are many different possible combinations of such vectors that will be apparent to the skilled man including an RNAi expression cassette targeting the host cell HPRT gene and a neomycin positive selection cassette.

The vectors and methods of the invention may comprise an RNAi that targets the positive selection marker on the gene targeting vector. The RNAi expression cassette may produce an RNAi that targets the positive selection marker such that cells that have randomly integrated the vector, or that include vectors present episomally, are negatively selected due to suppression of expression of the positive selection marker. The positive selection marker may be neo and the RNAi targeted to the neo gene. Other suitable markers include hygromycin and puromycin. The RNAi expression cassette may target a host cell gene that normally functions to inhibit homologous recombination in the host cell, such that homologous recombination is increased by expression of the RNAi. For example, the host cell gene may be in the cNHEJ, aNHEJ, homologous recombination, mismatch repair, or single strand DNA binding protein pathways. Suitable genes include DNAPKcs, MLH1, SH2, BLM, PCNA, RPA, POLD1, POLD2, POLD3, and POLD4. Following its introduction into the cell the transient expression of such an RNAi from the gene targeting vector functions to increase the number of homologous recombination events and thus increase the efficiency of the gene targeting process. When the targeting construct has been incorporated into the correct location in the host cell genome by a homologous recombination event the RNAi cassette is recombined away and down regulation of the host cell gene ceases.

This transient expression of the RNAi is important as continued disruption of the natural mechanisms of DNA repair could lead to mutations which could be detrimental to the cell. It could also lead to mutations that are not lethal, but might otherwise compromise the "isogenic" nature of the modified cells.

The vector may comprise a spacer (stuffer) DNA sequence adjacent to the RNAi expression cassette. The spacer DNA sequence is positioned between the ITRs or vector ends and the RNAi expression cassette. This may protect the RNAi expression cassette from being degraded through exonuclease activity within the cell. The spacer DNA sequence length may be varied depending on the type of vector used and space available but may, for example, be between about 10 and 100 nucleotides in length. As will be apparent to the skilled man, the vectors may comprise multiple RNAi expression cassettes, for example, at least one RNAi expression cassette adjacent to each of the vector ends or ITRs. These RNAi cassettes may be directed to the same target or may be directed to different targets. Possible combinations of RNAi cassettes include, for example, an RNAi that targets the positive selection cassette on the gene targeting vector and an RNAi that targets an endogenous host cell gene. One example of such a combination includes an RNAi targeting a neomycin positive selection marker on the vector and an RNAi targeting host cell HPRT. Another possible combination is a dual HPRT vector, which has an HPRT RNAi expression cassette at both ends of the vector.

The RNAi expression cassette may be shRNA, siRNA or miRNA. The construction and design of suitable RNAi expression cassettes is described in BMC Biotechnol. 2006 Jan 5;6:1. Design and cloning strategies for constructing shRNA expression vectors, Mclntyre GJ, Fanning GC. The small size of RNA expression cassettes is particularly useful in gene targeting vectors that have size constraints such as AAV. However, in general, it is desirable to keep the size of the RNAi expression cassette to a minimum to provide the maximum space for the targeting construct. When used in conjunction with a parvoviral vector having limited payload capacity, it is advantageous to incorporate a promoter of minimal size to drive the RNAi expression. A minimal U6 promoter has been identified which is substantially smaller in size and appears to provide suitable expression of an adjacent RNAi to allow for successful negative selection. For example, the shRNA cassette targeted to neo on the vector has a total length of 143bp, which comprises a minimal U6 promoter and an RNAi cassette targeting the neomycin gene.

The vectors of the invention may be "scarless" such that the targeting construct does not include a selection marker, for example, the targeting construct consists essentially of the genetic modification to be introduced flanked by regions of homology which are substantially identical to the genomic target locus, said regions each being at least 50bp in length.

The present invention also provides recombinant viruses comprising the viral vectors according to the invention, including recombinant parvoviruses.

The recombinant viruses may comprise a chimeric or modified capsid, including capsids that are designed to modify the tropism of the virus and/or enhance uptake of virus by a wide range of host cells.

The present invention also provides methods of using any of the vectors according to the invention and includes a method of producing a vertebrate cell having a genetic modification at a preselected genomic target locus, the method comprising: a) introducing into the vertebrate cell an effective amount of a gene targeting vector comprising, i) a targeting construct comprising the genetic modification

flanked by regions of homology which are substantially identical to the genomic target locus, said regions each being at least 50bp in length;

i) at least one parvoviral inverted terminal repeat (ITR) flanking the targeting construct;

ii) at least one RNAi expression cassette positioned

between the region of homology and the ITR;

v) optionally, a positive selection marker; and b) determining if the genomic target locus has the introduced genetic

modification.

The methods of the invention may also include the step of selecting for cells that have not incorporated the RNAi expression cassette, for example, by exposing the cell to a selection agent to negatively select cells that comprise the RNAi expression cassette. Suitable selection agents include, for example, HAT media when the RNAi targets HPRT.

The methods of the invention may further comprise the step of selecting for cells that have introduced the positive selection marker, for example, by exposing the cells to a selection agent. Suitable combinations of selection marker and selection agent are well known to the skilled man and include those discussed above.

The inventors have found that utilising the vectors and methods of the invention enhances the efficiency of the gene targeting process compared with vectors and methods that do not include the RNAi cassette. The methods of the present invention include methods wherein the targeting frequency is at least 3%.

In one embodiment the present invention includes a method of producing a vertebrate cell having a genetic modification at a preselected genomic target locus, the method comprising: a) introducing into the vertebrate cell an effective amount of a gene targeting

vector comprising, i. a targeting construct comprising the genetic modification flanked by regions of homology which are substantially identical to the genomic target locus, said regions being at least 50bp in length;

ii. at least one parvoviral inverted terminal repeat (ITR)

flanking the targeting construct;

iii. at least one RNAi expression cassette positioned

between the region of homology and the ITR, the

RNAi targeting a host cell gene such that cells that have randomly integrated the RNAi expression cassette, or that include vectors present

episomally, are sensitised to a selection agent; and iv. optionally, a positive selection marker; exposing the cell to the selection agent to negatively select cell that comprise the RNAi expression cassette; and

) optionally, selecting for cells that have introduced the positive selection marker. The host cell gene may be an endogenous gene such as HPRT, Thymidine Kinase, APRT or XGPRT that confers sensitivity to the selection agent or the host cell gene may be a heterologous gene that was previously introduced into the host cell to confer sensitivity to a selection agent. Surprisingly, the inventors have found that, when using the gene targeting vectors with a shRNA targeted to host cell HPRT, as described herein, an increase in gene targeting efficiency is seen even before the selection agent is added to negatively select cells that have reduced levels of HPRT due to shRNA production.

As will be apparent to the skilled man, the gene targeting vectors of the invention may be introduced into the cells by any suitable methods known in the art. The method selected is largely dependent upon the type of gene targeting vector selected, for example, plasmid vectors may introduced into the cell by any suitable techniques such as chemical transfection or electroporation. Viral gene targeting vectors, such as AAV, may,

advantageously, be introduced in the conventional manner as packaged virus so that they may enter the cell by viral transduction.

The methods and vectors of the invention can be used for introducing a second modification at a second target locus by using a second gene targeting vector according to the present invention. Additional target loci can be modified by transduction using gene targeting vectors that have appropriate targeting constructs.

Also provided by the invention are vertebrate cells that contain specific genetic

modifications at one or more preselected target loci that were introduced into the cells, or ancestors of the cells, by contacting the cells with a parvoviral vector that has a recombinant viral genome which includes a targeting construct that includes a DNA sequence which is substantially identical to the target locus except for the modification being introduced. These cells can be cultured in vitro, ex vivo, or can be part of an organism.

The vectors and methods of the invention can be used for targeting somatic cells or stem cells, including embryonic stem cells or induced pluripotent stem cells. The vectors and methods may be utilised in-vitro or ex-vivo.

In other embodiments, the invention provides methods for making a modification of a target locus in a cell in a vertebrate by administering the gene targeting viral vectors of the invention to the vertebrate. The invention also provides methods of making an animal that includes cells that have a modification of a target locus of interest. The methods involve introducing into a cell from which an animal can be reconstituted a gene targeting vector according to the present invention. Homologous pairing occurs between the targeting construct and the target locus resulting in the modification being introduced into the target locus. The cell and/or progeny of the cell is then allowed to develop into an embryo and brought to term. The resulting animals, which can be either transgenic or chimeric animals, are also part of the invention.

In other embodiments, the methods of the invention are used to obtain modified nuclei that are used in nuclear transplantation. These methods involve using the gene targeting methods of the invention to introduce a desired modification into a target locus in the genome of the cell that is to serve as the nucleus donor. The nucleus from a cell that has the desired modification is introduced into a second cell from which an animal can be reconstituted. This cell is then allowed to develop into an embryo and brought to term.

Again, the resulting animals, which are either transgenic or chimeric, are also provided by the invention.

The vectors and methods of the invention may, as is conventional in the art, include recombination signals such lox sites, e.g. to facilitate the removal of a positive selection cassette from a targeted cell.

Other methods provided by the invention for enhancing the efficiency of gene targeting involve treating a target cell with an agent that enhances targeting efficiency. These agents include, for example, one or more of a cell cycle modulator, a DNA repair modulator, a DNA recombination modulator, a modulator of chromatin packaging, an inhibitor of apoptosis, and a DNA methylation inhibitor. The agents may be small molecule inhibitors or RNAi. The methods of the invention include applying the agents to the target cell along with the vectors, or pre-treating the cells with agent before introducing the vectors.

The present inventors have found that, when targeting cells that are Mismatch Repair

Proficient (MMR), it may be beneficial to inhibit the MMR pathway by treating the cells with an agent that inhibits MMR (for example, an RNAi that targets a MMR pathway gene), or by including in the vector a RNAi expression cassette that targets an MMR gene. The MMR pathway inhibition may be transient. Suitable targets in the MMR pathway are known in the art. The inhibition of MMR (via treating with an agent or from an shRNA expressed from the vector) may be combined with a RNAi (for example shRNA) negative selection cassette in the vector (as described herein).

Thus a further method provided by the invention includes a method of producing a vertebrate cell having a genetic modification at a preselected genomic target locus, the method comprising:

a) treating a MMR proficient vertebrate cell with an agent that inhibits MMR;

b) introducing into the vertebrate cell an effective amount of a gene targeting vector comprising: i) a targeting construct comprising the genetic modification flanked by regions of homology which are substantially identical to the genomic target locus, said regions each being at least 50bp in length; and ii) at least one RNAi expression cassette positioned outside the region of homology, wherein the RNAi expression cassette is a negative selection cassette such that cells that have randomly integrated the RNAi expression cassette are negatively selected; and c) determining if the genomic target locus has the introduced genetic modification.

The gene targeting vectors according to the present invention may also be used in combination with one or more nucleases that are used to introduce either a single or double-strand break into a specific site. The use of nucleases in combination with the gene targeting vectors of the present invention to provide the donor molecule, should increase the frequency of homologous recombination events. However, the generation of single or double-strand breaks may be associated by a higher frequency of off-target events but the RNAi expression cassette on the vectors of the present invention enable easy selection or screening to remove cells which have incorporated the vector (donor) into random locations within the genome (off-targets). Examples of nucleases which can used in this embodiment include zinc finger nucleases (ZFNs), meganucleases, transcription activator like effector nucleases (TALENs), or the CRISPR/Cas9 system.

FIGURES

Figure 1: CDK2 D145N gene targeting vector with HPRT ShRNA negative selection cassette: Schematic depiction of the targeting vectors used in the example experiments to introduce the D145N point mutation into the CDK2 gene. Major features are shown along with their sizes in numbers of nucleotides(bp). (a) The single ShRNA negative selection cassette positioned at the end of the left homology arm is illustrated showing the HPRT ShRNA sequence, (b) The same vector as in (a) with the addition of a second HPRT ShRNA cassette positioned at the end of the right homology arm. ITR; internal tandem repeat, PGK;

phosphoglycerate kinase promoter, NEO; neomycin phosphotransferase gene, CDK2 exons (black boxes), LoxP (triangles), the CDK2 D145N point mutation is shown in exon 4.

Figure 2: HPRT ShRNA negative selection cassettes increases rAAV-mediated gene targeting frequencies: HCT116 cells were infected with rAAV vectors to target the CDK2 D145N mutation, vectors had either no ShRNA negative selection cassette, single HPRT or dual HPRT ShRNA cassettes. Infected cells were aliquoted by limiting dilution into 96-well plates and placed under G418 selection (0.3mg/ml) for the times indicated with or without subsequent lxHAT selection as indicated. The total number of correctly targeted clones was determined by diagnostic PCRs and is expressed as a percentage of the total clones screened. Figure 3: Variant designs of rAAV ShRNA negative selection vectors: Diagrams illustrate the different arrangements of ShRNA negative selection cassettes in rAAV vectors, including the orientation and positioning of one or more cassettes between the homology arms and ITR sequences:

a) shRNA expression 5'-3'; rest of sequence is homologous to endogenous target gene sequence;

b) shRNA expression 3'-5'; rest of sequence is homologous to endogenous target gene sequence;

c) shRNA expression is in either orientation; sequence between this and ITR is not homologous

but on other side is homologous;

d) shRNA expression is in either orientation; rest of sequence is NOT homologous to target gene. Figure 4A is a map of the targeting vector AAV-HPe3TNAsil71 that expresses a siRNA that can pair with sequences at bpl71 of the neo gene; the control vector AAV-HPe3TNAmU6 contains an empty mU6 cassette. Figure 4B shows that while 6TG-resistance rates (gene- targeted cells) were only slightly lower with the sil71 vector as compared to the control, there was a more than 10-fold drop in G418-resistance.

Figure 5 shows increased gene targeting frequency with an rAAV virus with dual shRNA HPRT cassettes targeting either the BRAF V600E mutation (Figure 5A) or the EGFR T790M mutation (Figure 5B). NT represents non-transfected. Figure 5C - MCFlOa a nd HCT116 cells (MMR deficient) were transfected with MSH2 siRNA, or left untransfected (NT) and cultured for 72 hours. Whole cell lysates were prepared and analysed by western blot for MSH2 protein expression.

EXAMPLES

Example 1 - rAAV gene targeting vector with shRNA targeting endogenous HPRT. Cell culture The human colon cancer cell line HCT116 was obtained from the American Type culture collection (ATCC) and maintained in RPMI 1640 media (Invitrogen) supplemented with 10% heat inactivated calf serum (Sigma), 2mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin (I nvitrogen). HEK293T cells were obtained from ATCC and cultured in DMEM F-12 Nutrient mix (HAM) (Invitrogen) supplemented with 10% heat inactivated calf serum, 100 U/ml penicillin and 100 U/ml streptomycin. For drug selection, the media was supplemented with G418 (0.3 mg/ml, Sigma) and lxHAT supplement (Invitrogen). All cell lines were grown at 37°C in a humidified incubator with 5% C0 ₂ .

Targeting vector construction and virus production

The targeting vectors described in figure 1 were generated by DNA synthesis of the homology arms and selection cassettes as designated (Genscript, NJ USA). The shRNA cassette Homo sapiens U6 promoter was obtained from Genbank (Genbank I D: JN255693.1) and the human HPRT ShRNA sequence obtained from reference 1. The synthesised fragment was cloned by restriction enzyme digestion and ligation into the pAAV-MCS backbone plasmid (Agilent) between the two copies of the AAV-2 ITR sequences to facilitate viral packaging.

Infectious rAAV was generated by co-transfection of the targeting vector and the pDG helper plasmid (PlasmidFactory GmbH, Germany) into HEK293T cells using lipofectamine LTX reagent (Invitrogen) following the manufacturer's protocol. Virus was harvested 72 hours after transfection. Briefly, media was collected from the T75 flask and the HEK293T cells were washed in 3 ml of phosphate-buffered saline (Invitrogen), 2 ml of TrypLE Express dissociation reagent (Invitrogen) was added to the flask which was incubated for 5 minutes at 37°C. Dissociated cells were harvested and the collected media and cell suspension centrifuged for 5 minutes at 1000 x g. Cell pellets and clarified supernatants were stored at - 80°C, before being subjected to three cycles of freeze and thaw. Each cycle consisted of 10 min freeze in a dry ice/ethanol bath, and 10 min thaw in a 37°C water bath. The lysate was then clarified by centrifugation at 1000 x g for 30 minutes. Approximately 2500 units of Benzonase nuclease (Sigma) was added to the clarified supernatant which was incubated at 37°C for a further 30 minutes. Virus was purified from the treated supernatant using the AAV Purification ViraKit (ViraPur, CA USA) according to the manufacturer's instructions. Aliquots of purified virus were stored at -80°C until use.

Virus titre and infection

The titre of purified viral stocks was measured by Q-PCR. Briefly, 5 μΙ of purified virus was treated with amplification grade DNase I (Sigma) for 30 minutes at 37°C, followed by treatment with proteinase K (Sigma) for 1 hour at 56°C. Dilutions of the treated virus were compared to dilutions of standard virus stocks (known titres) in Q-PCR assays using oligonucleotide primers and FAM-dye labelled probes (Applied biosystems) specific for the neomycin resistance selection cassette.

HCT116 cells were infected with purified virus at multiplicity of infection (MOI) of 100,000 genome copies/virus particles per cell. The appropriate volume of virus was diluted in culture media and added to cells plated in T175 flasks for 72 hours. Media was then replaced with media containing G418 for selection and infected cells were selected for two weeks. For HAT selection experiments, media was then replaced without G418 but with the addition of lxHAT supplement and cells cultured for a further seven days. After selection, cells were plating to single colonies across ten 96-well plates (1 cell/well). Colonies were allowed to expand without selection for a further two weeks until approximately covering >30% of the well surface before being harvested. Briefly, media was removed from the wells and cells were washed with 100 μΙ of phosphate-buffered saline. To each well, 30 μΙ of TrypLE Express dissociation reagent was added and the plates incubated for 5 minutes at 37°C before transfer of 5 μΙ of the cell suspension to 20 μΙ of DirectPCR lysis solution (Viagen). Cell lysates were then analysed by PCR.

PCR screening genomic DNA

Locus-specific targeting events were screened by PCR using a forward primer that was situated outside of the left homology arm (5'-CACCAGGCGGCTTTACTTAC-3') and a reverse primer (5'AGGTAGCCGGATCAAGCGTATGCAG-3') that was situated within the neomycin resistance cassette. All PCR reactions were performed with GoTaq Hot start Polymerase (Promega) using the conditions specified by the manufacturer. PCR reactions were performed in 15 μΙ total volumes in 96-well plates using the following cycling conditions: 1 cycle of 94°C for 3 minutes; 3 cycles of 94°C for 15 seconds, 64°C for 30 seconds, 70°C for 150 seconds; 3 cycles of 94°C for 15 seconds, 61°C for 30 seconds, 70°C for 150 seconds; 3 cycles of 94°C for 15 seconds, 58°C for 30 seconds, 70°C for 150 seconds; 35 cycles of 94°C for 15 seconds, 57°C for 30 seconds, 70°C for 150 seconds; 1 cycle of 70°C for 5 minutes. Amplified PCR products were resolved on 1% agarose gels by electrophoresis. Colonies harbouring a correctly targeted allele were scored positive based on the present of a unique PCR product of the correct molecular weight.

References

1. Guibinga GH, Hsu S and Friedmann T. Deficiency of the housekeeping gene

hypoxanthine-guanine phosphoribosyltransferase (HPRT) dysregulates neurogenesis. Mol Ther. 2010 Jan; 18(l):54-62.

Example 2 - rAAV gene targeting vector with shRNA targeting the positive selection cassette (neo).

The AAV-HPe3TNA vector used in this Example can disrupt exon 3 of H PRT by insertion of a TK promoter-neo cassette, conferring 6TG- and G418-resistance after gene targeting, but only G418-resistance after random integration. Typically, G418-resistance rates are 10-20 fold higher than 6TG-resista nce rates, reflecting the higher frequency of random integration (Hirata et al., 2002). This vector was modified by inclusion of a m urine U6 snRNA promoter (mU6) for siRNA transcription placed outside of the ta rgeting homology (Fig. 4A). Based on our prior analysis of randomly integrated AAV vector proviruses (Miller et al., 2002; Rutledge and Russell, 1997), we reasoned that most random integrants would contain the mU6 cassette, while gene-targeted cells would not. Thus, by including an siRNA cassette designed to inhibit neo expression, most random integrants would express the siRNA and not be G418-resistant.

The targeting vector AAV-HPe3TNAsil71 expresses a siRNA that can pair with sequences at bpl71 of the neo gene, and the control vector AAV-HPe3TNAmU6 contains an empty mU6 cassette (Fig. 4A). While 6TG-resistance rates (gene-targeted cells) were only slightly lower with the sil71 vector as compared to the control, there was a more than 10-fold drop in G418-resistance - see Figure 4B. I n fact the ratio of G418:6TG resistance was 1.2-4.5 for the sil71 vectors, as compared to 16-27 for the control. This represents a decrease in G418- resistant random integrants with the siRNA vector.

Example 3- I ncrease in gene targeting frequency by combination of MSH2 suppression and negative selection. MMR proficient cells (MCFlOa and NCI-H838) and MMR deficient cells (HCT116) were pre-treated with an agent that inhibits a gene in the MMR pathway by transfecting the cells with MSH2 siRNA, or left untransfected (NT). The cells were cultured for 72 hours before infection with a virus (Figure 5D) with a dual shRNA HPRT cassette targeting either the BRAF V600E mutation (Figure 5A) or the EGFR T790M mutation (Figure 5B). Cells were selected in G418 and HAT in combination for 2-weeks, then analysed for targeted allele frequency by locus specific ddPCR. NT represents non-transfected. Figure 5C - MCFlOa and HCT116 cells (MMR deficient) were transfected with MSH2 siRNA, or left untransfected (NT) and cultured for 72 hours. Whole cell lysates were prepared and analysed by western blot for MSH2 protein expression. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below. The term"cell line,"as used herein, refers to individual cells, harvested cells, and cultures containing the cells, so long as they are derived from cells of the cell line referred to. A cell line is said to be"continuous,""immortal,"or "stable" if the line remains viable over a prolonged time, typically at least about six months. To be considered a cell line, as used herein, the cells must remain viable for at least 50 passages. A"primary cell,"or"normal cell, "in contrast, refers to cells that do not remain viable over a prolonged time in culture.

The term"cis-active nucleic acid'Vefers to a nucleic acid subsequence that encodes or directs the biological activity of a nucleic acid sequence. For instance, cis-active nucleic acid includes nucleic acid subsequences necessary for modification of a nucleic acid sequence in a host chromosome, and origins of nucleic acid replication.

The term "exogenous" as used herein refers to a moiety that is added to a cell, either directly or by expression from a gene that is not present in wild-type cells. I ncluded within this definition of "exogenous" are moieties that were added to a parent or earlier ancestor of a cell, and are present in the cell of interest as a result of being passed on from the parent cell. "Wild-type" in contrast, refers to cells that do not contain an exogenous moiety.

"Exogenous DNA "as used herein, includes DNA sequences that have one or more deletions, point mutations, and/or insertions, or combinations thereof, compared to DNA sequences in the wild-type target cell, as well as to DNA sequences that are not present in the wild-type cell or viral genome.

The term "homologous pairing," as used herein, refers to the pairing that can occur between two nucleic acid sequences or subsequences that are complementary, or substantially complementary, to each other. Two sequences are substantially complementary to each other when one of the sequences is substantially identical to a nucleic acid that is complementary to the second sequence, as defined below.

The term "host cell" or "target cell" refers to a cell to be transduced with a specified vector. The cell is optionally selected from in vitro cells such as those derived from cell culture, ex vivo cells, such as those derived from an organism, and in vivo cells, such as those in an organism.

The term "identical" in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. Optimal alignment of sequences for comparison can be conducted, e. g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J.

Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics

Computer Group, 575 Science Dr., Madison, Wl), or by inspection.

An indication that two nucleic acid sequences are "substantially identical" is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. Another indication that two nucleic acid sequences are substantially identical is that the two molecules and/or their complementary strands hybridize to each other under stringent conditions. The phrase"hybridizing specifically to'Vefers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e. g., total cellular) DNA or RNA. The

term"stringent conditions'Vefers to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid

concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium). Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 C for short probes (e. g., 10 to 50 nucleotides) and at least about 60 C for long probes (e. g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Specific hybridization can also occur within a living cell.

An"inducible"promoter is a promoter which is under environmental or developmental regulation.

The term "labeled nucleic acid probe" refers to a nucleic acid probe that is bound, either covalently, through a linker, or through ionic, van der Waals or hydrogen "bonds" to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.

The term "label" refers to a moiety that is detectable by spectroscopic, radiological, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32p, 35S, fluorescent dyes, electron-dense reagents, enzymes (e. g., as commonly used in an ELISA), biotin, dioxigenin, green fluorescent protein (GFP), or haptens and proteins for which antisera or monoclonal antibodies are available. The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single-or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof.

The term "operably linked" refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs

transcription of the nucleic acid corresponding to the second sequence.

The term "recombinant parvoviral vector genome" refers to a vector genome derived from a parvovirus that carries non-pa rvovira I DNA in addition to parvoviral viral DNA. The recombinant vector genome will typically include at least one targeting construct.

The term "replicating cell" refers to a cell that is passing through the cell cycle, including the S and M phases of DNA synthesis and mitosis. The term "subsequence" in the context of a particular nucleic acid sequence refers to a region of the nucleic acid equal to or smaller than the specified nucleic acid.

A "target locus"as used herein, refers to a region of a cellular genome at which a genetic modification is desired. The target locus typically includes the specific nucleotides to be modified, as well as additional nucleotides on one or both sides of the modification sites.

A "targeting construct" refers to a DNA molecule that is present in the recombinant parvoviral vectors used in the methods of the invention and includes a region that is identical to, or substantially identical to, a region of the target locus, except for the modification or modifications that are to be introduced into the host cell genome at the target locus. The modification can be at either end of the targeting construct, or can be internal to the targeting construct. The modification can be one or more deletions, point mutations, and/or insertions, or combinations thereof, compared to DNA in the wild-type target cell.

The term "transduction" refers to the transfer of genetic material by infection of a recipient cell by a recombinant viral vector.

A cell that has received recombinant parvoviral vector DNA, thereby undergoing genetic modification, is referred to herein as a"transduced cell,"a"transfected cell,"a"modified cell, "or a "recombinant cell" as are progeny and other descendants of such cells.

The term "transgenic cell" refers to a cell that includes a specific modification of the cell's chromosomal or other nucleic acids, which specific modification was introduced into the cell, or an ancestor of the cell. Such modifications can include one or more point mutations, deletions, insertions, or combinations thereof. When referring to an animal, the term "transgenic" means that the animal includes cells that are transgenic. An animal that is composed of both transgenic cells and non-transgenic cells is referred to herein as a "chimeric" animal.

The term "vector" refers to an agent for transferring a nucleic acid (or nucleic acids) to a host cell. A vector comprises a nucleic acid that includes the nucleic acid fragment to be transferred, and optionally comprises a viral capsid or other materials for facilitating entry of the nucleic acid into the host cell and/or replication of the vector in the host cell (e. g., reverse transcriptase or other enzymes which are packaged within the capsid, or as part of the capsid). The term "viral vector" refers to a vector that comprises a viral nucleic acid and can also include a viral capsid and/or replication functions.

As used herein, the term "stem cell" refers to an undifferentiated cell that can be induced to proliferate. The stem cell is capable of self-maintenance or self-renewal, meaning that with each cell division, one daughter cell will also be a stem cell. Stem cells can be obtained from embryonic, post-natal, juvenile, or adult tissue. Stem cells can be pluripotent or multipotent. The term "progenitor cell," as used herein, refers to an undifferentiated cell derived from a stem cell, and is not itself a stem cell. Some progenitor cells can produce progeny that are capable of differentiating into more than one cell type.

As used herein, the term "genetic modification" refers to a permanent or transient genetic change induced in the genome of a cell following introduction of new nucleic acid (i.e., DNA exogenous to the cell). Genetic modification can be accomplished by incorporation of the new ("exogenous") DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. Genetic modifications include, e.g., duplication of an endogenous nucleotide sequence in the nuclear genome. Following induction of a genetic change in the genome (e.g., the nuclear genome) of a cell, the exogenous DNA can remain in the cell (e.g., can be integrated into the genome, or can be present extrachromosomally), or can be absent from the cell (e.g., deleted from the genome). Means for effecting a "genetic modification" exclude parthenogenesis.

A "parvoviral vector" refers to a vector based on or derived from a parvovirus such as adeno-associated virus (AAV) and minute virus of mice (MVM). See, e.g., Hendrie et al. (2003) J. Virol. 77:13136-13145; and Russell et al. (2002) Nat. Biotech. 20:658.

"AAV" is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector"). The term "AAV" includes, but is not limited to, AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), and AAV type 8 (AAV-8).

An " rAAV vector" as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell. I n general, the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

An "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild- type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector". Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle. "Packaging" refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.

A "helper virus" for AAV refers to a virus that allows AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein- Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

"Helper virus function(s)" refers to function(s) encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus function" may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans. An "infectious" virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is trophic. The term does not necessarily imply any replication capacity of the virus. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the P:l ratio, or the ratio of total viral particles to infective viral particles.

The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc. A "therapeutically effective amount" or "efficacious amount" means the amount of a compound or a number of cells that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on one or more factors such as the cell, the disease and its seventy and the age, weight, etc., of the subject to be treated.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an HLA homozygous cell" includes a plurality of such cells and reference to "the rAAV vector" includes reference to one or more rAAV vectors and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a

"negative" limitation.