BIOLOGICAL MATERIALS AND USES THEREOF

The present invention relates to methods of introducing nucleic acid molecules into spermatozoa so as to obtain transgenic spermatozoa, and methods of using the transgenic spermatozoa so obtained in transgenic animal reproduction.

The field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and phenomena of gene activation, expression, and interaction. This technology has been used to produce models for various human diseases and diseases of other animals. Transgenic technology is amongst the most powerful tools available for the study of genetics, and the understanding of genetic mechanisms and function. It is also used to study the relationship between genes and diseases. About 5,000 diseases are caused by a single genetic defect. More commonly, other diseases are the result of complex interactions between one or more genes and environmental agents, such as viruses or carcinogens. The understanding of such interactions is of prime importance for the development of therapies, such as gene therapy and drug therapies, and also treatments such as organ transplantation. Such treatments compensate for functional deficiencies and/or may eliminate undesirable functions expressed in an organism. Transgenesis has also been used for the improvement of livestock, and for the large-scale production of biologically active pharmaceuticals. Transgenic animals may also be generated in order to provide organs suitable for xenotransplantation.

The classic method for generating transgenic animals developed by Gordon and Ruddle in 1980 (Gordon IW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH (1980) Proc Natl Acad Sci USA 77: 7380-7384), is the microinjection of the gene of interest into the nucleus of a fertilised egg and the subsequent transfer of the injected embryos into a foster mother. About 0.1-1.0% of these injected eggs result in a transgenic animal. This method is routine in mice as can be seen by the thousands of existing transgenic lines. By contrast, large transgenic animals are difficult to generate using the classic technique due to the inherent inefficiency of the technique, the small number of fertilised eggs that can be harvested from large animals even after super ovulation, and underdeveloped Artificial Reproductive Technologies (ART) in many species.

Larger animals would be much more suitable than small animals to study the effects and treatment of most human diseases because of their greater similarity to humans in many aspects such as physiology, and also the size of their organs. Now that transgenic animals with the potential for human xenotransplantation are being developed, larger animals, of a size comparable to man will be required. Transgenic technology will allow that such donor animals will be immunocompatible with the human recipient. Historical transgenic techniques, however, require that there be an ample supply of fertilized female germ cells or eggs. Most large mammals, such as primates, cows, horses and pigs produce only 10-20 or less eggs per animal per cycle even after hormonal stimulation. Consequently, generating large animals with these techniques is prohibitively expensive.

To improve the rate of transgenesis in larger mammalian species and birds, researchers have attempted to develop alternative methods based on introduction of nucleic acid molecules into male germ cells (Smith and Spadafora, (2005) BioEssays 27:551-562). Generally, vast numbers of male germ cells are available for such techniques. Most male mammals generally produce at least 10 ⁸ spermatozoa (male germ cells) in each ejaculate. This is in contrast to only 10-20 eggs in a mouse even after treatment with superovulatory drugs. A similar situation is true for ovulation in nearly all larger animals. For this reason alone, male germ cells have been an attractive target for introducing foreign DNA into the germ line.

In one such method, seminal plasma-free sperm cells are suspended in an appropriate medium and incubated with DNA. The resultant DNA-carrying sperm are then used to fertilize eggs, via in vitro fertilization (IVF) or artificial insemination (Al). However, transgenes may not be integrated or maintained in animals developed from the fertilized eggs, or the progeny of such animals and, in any case, Artificial Reproductive Technologies (ART) are underdeveloped in many species. This method has not been reproduced by other groups.

Other methods have been developed which involve in vitro transduction/incubation of DNA with male germ stem cells (i.e. immature male germ cells) followed by transplantation of modified cells back into the testis of recipients, in vivo injection of viruses/liposome coated DNA into the testis, or

electroporation of DNA into the testis. A potential advantage of such methods is that animals harbouring the transgenic sperm cells can be mated naturally and do not require further intervention or ART.

Sato, Ishikawa and Kimura (MoI. Reprod. Dev. (2002) 61:49-56) described a method of introducing a DNA construct encoding a fluorescent marker protein (enhanced GFP) into the testis. The fluorescent marker gave rise to weak or no fluorescence unless the DNA was incorporated into cells of the testis by electroporation. The transgene was found to be incorporated in spermatozoa obtained from the epididymis. However, the eGFP marker did not give rise to fluorescence in these cells.

Sciamanna et al (Biochem. Biophys. Res. Comm. (2003) 312:1039-1046) described a method of introducing a ribonucleic acid molecule encoding a beta-galactosidase gene into spermatozoa obtained from the epididymis. The ribonucleic acid was reverse transcribed to cDNA and propagated as an extrachromosomal cDNA in founder and Fi progeny. The ribonucleic acid molecule used was purified from retroviral particles bearing a vector described in WO 99/25862 to Nature Technology, Inc. The ribonucleic acid molecule does not encode a reverse transcriptase and therefore the reverse transcriptase activity relied upon by the method of Sciamanna et al is endogenous to the spermatozoa. This DNA was not integrated into the genome of the spermatozoa.

In WO 99/25863, WO 00/29601 and WO 00/69257 to Cedar-Sinai Medical Center and Imperial College, incorporated herein by reference, retroviral vectors were used to introduce a gene of interest into immature male germ cells in vitro or in vivo. In the in vivo method, the viral vectors were introduced into the seminiferous tubules of the testis either by direct injection or via the Rete testis or vas efferens. After recovery from surgery and maturation of the transduced germ cells, the male may be mated naturally or sperm collected for artificial insemination (Al). In the in vitro method, immature male germ cells were exposed to the viral vectors in vitro and then introduced into the testis of a recipient male. After maturation of the introduced transduced germ cells, the male may be mated naturally or sperm collected for artificial insemination (Al).

Mature spermatozoa are understood to contain tightly packed chromatin (condensed chromatin) so as to maximise efficient transfer to the oocyte during fertilisation (see e.g. Essential Reproduction, Johnson and Everitt 4 ^th Ed, 1995 pages 143-145). Such condensed chromatin has previously been considered incapable of incorporating foreign nucleic acid molecules, such as nucleic acids carried by a retrovirus, to produce transgenic spermatozoa. This is because the combined effect of the condensed nature of the chromatin and the tough outer layers of spermatozoa meant that the DNA was not accessible to carriers such as viruses (see Histological and histopathological evaluation of the testis. Russell, Ettlin, SinhaHikim and Clegg. 1990. Cache River Press).

The inventors have devised methods of introducing a nucleic acid molecule into a mature spermatozoan to obtain a transgenic spermatozoan. Such transgenic spermatozoa may be used to obtain transgenic zygotes by fertilization of a female germ cell (also known as oocyte, egg or ovum). Hence, transgenic animals can be obtained by use of the method of the invention.

A first aspect of the invention provides a method of incorporating a nucleic acid molecule of interest into a mature spermatozoan to obtain a transgenic spermatozoan comprising exposing a mature spermatozoan of a vertebrate to a pseudotyped viral vector comprising a nucleic acid molecule of interest under conditions which allow for the introduction of said nucleic acid of interest into the spermatozoan. Preferably the nucleic acid molecule includes a transcription unit.

By "introduction" we mean the nucleic acid of interest is taken up into the interior of the cell and preferably integrated into the genome of the spermatazoan.

By "mature spermatozoa" we mean haploid male germ cells that lack cytoplasmic connections with immature germ cells or somatic cells. Preferably, they are free swimming and motile and are capable of targetting and penetrating the oocyte. Their nucleus is able to fuse naturally (i.e. without external assistance) with the nucleus of the oocyte to form a zygote. Such mature spermatozoa can be located in the seminiferous tubules, the rete testis, the vasa, the epididymis, the vas deferens and also in semen (both ejaculated and pre-ejaculated). Such mature spermatozoa may also be found in the female genital tract after mating.

Mature spermatozoa are also known as sperm and the terms "mature spermatozoa", "spermatozoa", "mature sperm" and "sperm" are used interchangeabley herein.

By "transgenic" we mean that a foreign nucleic acid molecule has been introduced into the cell. Generally, the foreign nucleic acid molecule is integrated into the DNA or chromosomes of the cell. The present invention is applicable to the production of transgenic animals containing foreign genetic material, i.e., exogenous, transgenic genetic material from the same species, or material from a different species, including biologically functional genetic material. The foreign nucleic acid molecule may also contain regions from prokaryotic organisms, such as bacteria, or viruses. In other instances, the genetic material is allogeneic genetic material, obtained from a different strain of the same species, for example, from an animal having a normal form of a gene, or a desirable allele thereof. Also the nucleic acid molecule may be a hybrid construct consisting of a promoter and a coding region linked together. The promoter and the coding region may be obtained from different species or from regions of genetic material present in the same species, which are not normally juxtaposed. The foreign nucleic acid molecule may contain a region, the nucleic acid sequence of which has been designed by man.

Spermatozoa are highly differentiated cells adapted to transport paternal DNA to the oocyte. The spermatazoa arise in the seminiferous tubules of the testis through a process of spermatogenesis which can be divided into three phases, namely spermatocytogenesis, meiosis and spermiogenesis. In spermatocytogenesis, spermatogonia divide, producing successive generations of cells that finally give rise to spermatocytes. In meiosis, spermatocytes go through two successive divisions, with a 50% reduction in the number of chromosomes and amount of DNA per cell, producing spermatids, which are haploid cells. In spermiogenesis, spermatids undergo an elaborate process of cytodifferentiation, producing spermatozoa.

Spermatids are located juxtaluminally within the seminiferous tubule and are closely associated with somatic Sertoli cells, where they differentiate in a process that includes formation of the acrosome, condensation and elongation of the nucleus, development of the flagellum and the loss of- much of the cytoplasm.

During division of the spermatogonia, the resulting cells do not separate completely but remain attached by cytoplasmic bridges. The sloughing of the cytoplasm and cytoplasmic bridges as residual bodies by spermatids leads to the separation of the spermatids from less mature germ cells.

Finally, mature spermatozoa are released into the lumen of the seminiferous tubule, are transported to the epididymis and then to the vas deferens where they may be recovered from any of these vessels. They can also be recovered from the genital tract of the female after mating.

Mature spermatozoa may also be recovered without the requirement for a surgical procedure, from semen produced by a male vertebrate. For example, semen may be obtained by electro-ejaculation or suitable artificial stimulation (masturbation). Alternatively, mature spermatozoa may be recovered directly from the epididymis. Mature sperm can be retrieved without sacrifice by an open epididymal sperm aspiration (OESA), which is a minimally invasive surgical technique for sperm retrieval (Lania C, Grasso M, Fotuna F, De Santis L, Fusi T (2006) Arch Esp urol 59:313-316. Open epididymal sperm aspiration (OESA): minimally invasive surgical technique for sperm retrieval). Finally, mature sperm can also be obtained by sacrificing the animal, dissecting and puncturing the epididymis and allowing mature sperm swim up a needle or tube or, alternatively, by homogenization of the whole testis. Sperm may also be retrieved from the female genital tract after mating by flushing and aspiration.

Preferably the pseudotyped viral vector is selected from any viral vector capable of transgenesis, including retroviruses, adeno-associated virus and herpesvirus.

Most preferably the virus vector is a pseudotyped retroviral vector.

One advantage of the method of the first aspect of the invention is that transgenic mature spermatozoa may be obtained by exposing mature spermatozoa to a pseudotyped viral vector. As the transgenic spermatozoa do not need to undergo further stages of development within the testis of the male vertebrate, a transgenic spermatozoan obtained by the method of the first aspect of the invention may be used to fertilize the oocyte directly in vitro by in vitro fertilisation (IVF) or by injection of the spermatozoa into the oocyte by intracytoplasmic sperm

injection (ICSI), without being introduced into a male animal. The fertilised oocyte is then introduced into the uterus or suitably-prepared female. Thus, the method of the first aspect of the invention may include the additional step of using the transgenic spermatozoan to fertilize a female germ cell, with the limitation that the transgenic spermatozoan is not first introduced into a male animal.

Another advantage of the invention is that a pseudotyped viral vector comprising the nucleic acid molecule of interest is used to incorporate the nucleic acid molecule of interest into a spermatozoan.

Retroviruses are single-stranded RNA viruses. The RNA may be contained within a virus particle or virion. During an infection process in a host subject, RNA viruses are reverse transcribed into double-stranded DNA. The DNA is incorporated into the cellular DNA of the host by a process of non-homologous recombination. The integrated proviral DNA is capable of expressing viral RNA as well as RNA encoding the proteins that make up the virion. Retroviral vectors of the invention may be used in a form in which the genetic material is in the form of RNA or DNA. Typically, an RNA form is used, most typically, a single-stranded RNA form.

Retroviral vectors suitable for use in the invention may be obtained commercially or may be designed according to the principles set out in "Principles of Retroviral Vector Design" in Retroviruses, Coffin JM, Hughes SH and Varmus HE, 1997, Cold Spring Harbor Laboratory Press (incorporated herein by reference). A retroviral vector may be replication-competent or replication-defective. Preferably, the retroviral vector is replication-defective. A typical replication- defective retroviral vector comprises a RNA molecule bearing cis-acting vector sequences needed for transcription, reverse-transcription, integration and translation. For replication-defective viruses, packaging of viral RNA into viral particles is done by helper producer cells which express the trans-acting retroviral gene sequences (as proteins) needed for production of virus particles. By separating the cis- and trans-acting vector sequences completely, the virus is unable to maintain replication for more than one cycle of infection. Such a viral particle is conventionally referred to as being "replication-defective".

The viral vector for use in the invention is preferably pseudotyped. By "pseudotyped", we include the meaning that the virus particles containing the genetic material of the vector have a viral coat protein which is not the viral coat protein naturally occurring in a virus.

The concept of pseudotyping first arose in the field of virology in 1980. When a host cell was infected by two distinct enveloped viruses, the genome of one virus produced from the host cell was subsequently found to contain either its own envelope, the envelope of the unrelated virus which also infected the host cell, or a mixture of both the envelopes. See Zavada J (1982) The pseudotypic paradox. J Gen Virol 63, 15-24; Cronin J, Zhang XY, Reiser J (2005) Curr Gene Ther. Aug; 5(4):387-98. Review. Erratum in: Curr Gene Ther. 2005 Oct; 5(5):531. Altering the tropism of lentiviral vectors through pseudotyping.

The viral coat protein contributes to, or is responsible for, determining which cells can be infected by the virus e.g. a retrovirus.

The viral coat can also be an engineered envelope protein also commonly referred to as a chimeric envelope, i.e. a coat with a modified binding site, for example gamma retroviral envelopes containing modifications to their binding sites, the Haemaglutinin Antigen (HA) of the influenza virus coat protein or VSV pseudotypes containing novel binding sites within the envelope (Verhoeyen E and Cosset FL (2004) Surface-engineering of lentiviral vectors J Gene Med Feb 6 Suppl 1 : S83-94).

Preferably, the viral vector is pseudotyped (preferably a pseudotyped retrovirus) to allow efficient infection of spermatozoa of a vertebrate of the species to which the method is to be applied.

Examples of a retroviral vector system that can be used for introducing transgenic material into a mature sperm are described in the following examples and other methods and viruses are described in WO 2004/007735 (Imperial College Innovations Limited).

In the method of the invention, the viral vector comprises a nucleic acid molecule of interest. When the genetic material of the vector is incorporated into the

mature spermatozoan, the nucleic acid molecule of interest is therefore also incorporated. When using a retrovirus, the genetic material of the vector, including the nucleic acid molecule of interest, is integrated into the germ line of the mature spermatozoan. The nucleic acid molecule of interest may be located within the retroviral vector genome as described in "Principles of Retroviral Vector Design" (supra). The nucleic acid molecule of interest may be operatively linked to the promoter in the retroviral long terminal repeat (LTR). Changes can be made in the enhancer/promoter of the LTR to provide tissue-specific expression or inducibility. Alternatively, a single coding region can be expressed using an internal promoter, which allows more flexibility in promoter selection. Where expression of more than one gene is required, internal ribosome entry sites (IRES) may be used to allow translation of multiple coding regions from a single mRNA. Alternatively, different proteins may be expressed from alternatively spliced mRNAs transcribed from one promoter or transcription of different genes may be driven by different promoters. Suitable promoters include tissue specific promoters, spermatozoan specific promoters, constitutive promoters and/or inducible promoters.

In a preferred embodiment of the first aspect of the invention, the pseudotyped viral vector contains a selectable marker capable of conferring an altered (selectable) phenotype on the transgenic spermatozoan. The viral vector genome may be introduced into packaging cells by cotransfection with a separate plasmid comprising a gene encoding the selectable marker. In this scenario, the selectable marker is expressed in the viral vector budded from the packaging cells, but is not encoded in the genome of the viral vector. Thus, the selectable marker gene is not introduced into the mature spermatozoan. Alternatively, the viral vector genome may contain a gene encoding the selectable marker. The selectable marker gene may be a nucleic acid molecule that is the nucleic acid molecule of interest, or it may be an additional nucleic acid molecule. In this scenario, the gene encoding the selectable marker is introduced into the mature spermatozoan. Typically, the genetic material of the vector, including the gene encoding the selectable marker, is integrated into the germ line of the mature spermatozoan.

Suitably, where a gene encoding the selectable marker is introduced into the spermatozoan, the method of the first aspect of the invention further comprises

the step of allowing the transgenic spermatozoan to express the selectable marker. The selectable marker may be expressed after the gene has integrated into the genome. Alternatively, the gene may be expressed from viral RNA, or DNA reverse-transcribed from the viral RNA, which is present within the cytoplasm. The viral RNA or DNA reverse-transcribed from the viral RNA may subsequently integrate into the genome.

Suitably, the selectable marker is a fluorescent protein, a membrane-anchored protein or a protein which confers resistance to a toxin on the transgenic spermatozoan. Suitably, the selectable marker is encoded by, for example, a bacterial neomycin gene which confers resistance to G418; a bacterial hygromycin phosphotransferase gene which confer resistance to hygromycin; a mutant mouse dihydrofolate reductase gene which confers resistance to methotrexate; the multidrug resistance gene (mdr) which confers resistance to a variety of drugs; or a bacterial gene which confers resistance to puromycin or phleomycin, Suitable fluorescent proteins useful as selectable markers are Green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (EGFP), Yellow Fluorescent Protein, Blue Fluorescent Protein, Cherry Fluorescent Protein, a phycobiliprotein, such as phycoerythrin or phycocyanin. A membrane- anchored protein is a protein which is tethered to the plasma membrane of a cell and has an extracellular portion which is a ligand for a suitable detection reagent. For example, the extracellular portion may be a ligand for an antibody, which may be used to detect expression of the membrane-anchored protein by the cell. Membrane-bound EGFP is a suitable membrane-anchored protein. Alternatively, the selectable marker may be a luminescent protein.

Preferably, the method contains the further step of selecting the transgenic spermatozoan based on an altered phenotype conferred by the selectable marker. Where the selectable marker is a fluorescent protein, the selection may be performed by a fluorescence activated cell sorter. Where the selectable marker confers resistance to a toxin, transgenic spermatozoa resistant to the toxin could be positively selected. Preferably, positive selection is performed in vitro. Where the selectable marker is a membrane-anchored protein, a suitable detection reagent may be used to bind to the membrane-anchored protein. Where the detection reagent is fluorescent, selected may be performed by a fluorescence activated cell sorter. It will be appreciated that cells may also be

sorted where the detection reagent is not itself fluorescent, but is bound by a suitable fluorescent secondary reagent. Alternatively, selection may be performed by capture on paramagnetic beads coated with a binding reagent which either binds directly to the membrane-anchored protein, or binds to the detection reagent which is bound to the membrane-anchored protein. For example, the membrane-anchored protein may be bound by a biotinylated antibody, which may itself be bound by streptavidin-coated paramagnetic beads. Following capture of the cells on the paramagnetic beads, the beads are captured in a magnetic field. The cells may be released from the beads without damage to the cells.

Routine selection of cells with paramagnetic beads (commercially available from Dynal or Miltenyi) can be conducted by either disassociating or not disassociating the antibody-bead complex. In some instances cells selected in this method can be utilised directly following magnetic capture in downstream applications such as differentiation and transplant related applications without the need to dissassociate. Alternately if disassociation is required by the circumstances, the cells can be released from the beads by removal from the magnetic field and subsequent treatment using chemicals or changes in pH known to disassociate the beads leaving the selected cells intact.

It is a surprising feature that the selectable marker may be expressed by the transgenic spermatozoan. Mature spermatozoa are not thought to be competent to express exogenous genes. For example, Sato, Ishikawa and Kimura (MoI. Reprod. Dev. (2002) 61:49-56) describe the introduction of a DNA construct encoding eGFP into the testis of a mouse. Even though the eGFP construct was incorporated into spermatozoa, it was not expressed by the spermatozoa. However, it was expressed by neighbouring epithelial cells in the epididymis.

It is an advantage of the present invention that transgenic spermatozoa may be detected by virtue of their expression of a selectable marker. The expression of the selectable marker gives a rapid indication that the method of the first aspect of the invention has been performed successfully. This feature can be used to select mature transgenic spermatazoa expressing the selectable marker for use in other embodiments of the invention. Alternatively, where the mature spermatozoa are exposed to the retroviral vector in the male reproductive organs,

a sample of the mature spermatozoa may be collected to test for expression of the selectable marker. Where the sample is positive, the male may be mated naturally to give rise to transgenic progeny.

Where the pseudotyped viral vector contains a selectable marker, the first aspect of the invention may include the additional step of obtaining a sample of the transgenic spermatozoa and examining for expression of a selectable marker.

The pseudotyped viral vector may be administered into a vas deferens or epididymis of the vertebrate allowing for the exposure of the spermatozoan to the pseudotyped viral vector. The epididymis is a narrow, tightly-coiled tube connecting the rear of the testis to the vas deferens. The epididymis consists of three parts: head, body and tail. Spermatozoa are stored in the head of the epididymis, from which they enter the body of the epididymis, which is a highly convoluted duct, in which final maturation of the spermatozoa takes place. In particular, epithelial cells participate in the uptake and digestion of residual bodies that are eliminated during spermiogenesis. The tail of the epididymis is continuous with the vas deferens, which is the secretory duct of the testis. Spermatozoa therefore pass through the vas deferens from the epididymis during ejaculation. Where the viral vector is administered to the epididymis or the vas deferens, it will be encountered by mature spermatozoa. Methods of introducing material into the epididymis or vas deferens are known in the art. For example, Sato, Ishikawa and Kimura (MoI. Reprod. Dev. (2002) 61:49-56) describe a method in which a solution was injected into a mouse testis, from where it rapidly appeared in the epididymis. Huguet and Esponda (MoI. Reprod. Dev. (1998) 51 :42-52) describe a method in which a solution was injected into the vas deferens of a rat or a mouse.

Where the viral vector is administered to the epididymis or the vas deferens, a sample containing transgenic spermatozoa may be collected from the vertebrate using OESA, by ejaculation or by sacrificing the animal and releasing the mature spermatozoa from the epididymis or vas deferens.

As a preferred alternative to administering the viral vector to the epididymis or the vas deferens, mature spermatozoan may be exposed to the pseudotyped viral vector in vitro.

Suitably, where spermatozoa are exposed to the pseudotyped viral vector in vitro to obtain transgenic spermatozoa, or where transgenic spermatozoa are collected after administration of the pseudotyped viral vector to the epididymis or vas deferens, the method of the first aspect of the invention may includes a further step of cryopreserving the transgenic spermatozoa. Methods of cryopreserving cells are known in the art and are also disclosed in, for example, WO 99/25863. Cryopreservation may be used where it is desired to use the transgenic spermatozoa at a remote location from where they are generated. For example, it may be desirable to transport the transgenic spermatozoa to establish transgenic progeny at other locations. This approach is also applicable to the preservation of endangered species.

It is preferred that, in the method of the first aspect of the invention, a retroviral vector is pseudotyped with a gamma retroviral envelope such as Amphoteric, GALV or RD114, or a non-retroviral envelope such as VSV-G (Cronin J, Zhang XY, Reiser J (2005). Curr Gene Ther. Aug; 5(4); 387-98. Review. Erratum in: Curr. Gene Ther. 2005 Oct; 5(5); 531. Altering the tropism of ientiviral vectors through pseudotyping). Retroviral vectors pseudotyped with VSV-G envelope have a broad specificity for different host cells. Other examples of pseudotyping are:

Preferably, the pseudotyped viral vector also comprises a molecule which targets the viral vector to a mature spermatozoan. Suitably, the method of WO 2004/007735 to Imperial College Innovations Ltd (incorporated herein by reference) is used to provide a retroviral vector with a suitable targeting molecule. WO 2004/007735 describes a method of making viral particles having a modified cell binding activity. Viral particles are obtained by budding from a viral packaging cell. The viral packaging cell contains viral nucleic acid encoding a viral particle, and nucleic acid encoding a targeting molecule. When the viral particle buds from the packaging cell membrane, the targeting molecule is incorporated into the viral particle to modify its cell binding activity. Typically, the targeting molecule is a polypeptide, most typically a membrane-bound polypeptide. Non-membrane bound polypeptides may be used if the polypeptides are fused to a region of polypeptides that have a membrane binding region. The targeting molecule may be an antibody or an antigen binding fragment thereof. For example, the targeting molecule may contain an antigen binding fragment that is capable of binding selectively to mature spermatozoa. Envelope proteins engineered to redirect their binding to new cell surface molecules, by introducing N-terminal extensions, insertions, or replacements to regions of the envelope protein are known (Russell SJ, Cosset FL (1999).

Modifying the host range properties of retroviral vectors. J Gene Med 1: 300- 311 ). Suitable modifications are incorporation of growth factor-binding regions or the addition of single-chain antibodies).

In a particularly preferred embodiment, the targeting molecule is c-kit ligand. c-kit ligand (also known as stem cell factor (SCF)) is a ligand that binds to the c-kit receptor protein found on the surface of a variety of cell types. Recently, c-kit receptor has been found to be expressed on the acrosome of mature spermatozoa (Feng HL, Sandlow Jl, Zheng LJ (2005), c-kit receptor and its possible function in human spermatozoa MoI Reprod Dev. 70(1):103-10; Feng H, Sandlow Jl, Sandra A (1997). Expression and function of the c-kit proto- oncogene protein in mouse sperm. Biol. Reprod. JuI; 57(1): 194-203). WO 2004/007735 describes how c-kit ligand may be incorporated into the membrane of a viral particle, c-kit ligand exists in two forms, a longer soluble form and a shorter membrane bound form. The two forms result from differential mRNA splicing, with the soluble form having a proteolytic cleavage site encoded by exon 6, while the membrane bound form has no exon 6 and thus no proteolytic cleavage site. The membrane bound form is particularly suitable as a targeting molecule in the method of the first aspect of the invention.

A suitable targeting molecule may be used to increase the efficiency of infection of a mature spermatozoan compared to other cell types which do not express on their surface a ligand of the targeting molecule. Advantageously, in the method of the first aspect of the invention in which the pseudotyped viral vector comprising the targeting molecule is administered to the epididymis or vas deferens of the vertebrate, the pseudotyped viral vector will infect mature spermatozoa, but will not infect other cells encountered in the vertebrate, or will do so at only very low frequency. Ideally, in this embodiment of the invention, no cells other than mature spermatozoa will be infected by the pseudotyped viral vector. Thus, following ejaculation of the transgenic spermatozoa, the vertebrate does not carry the viral vector and therefore it is not at risk of viral genetic material recombining with endogenous viral genetic material or infecting viruses to produce a replication competent virus.

WO 2004/007735 describes that an ecotropic retrovirus did not transduce the human cell line Mo7e in vitro, but ecotropic retrovirus modified to include

membrane-bound SCF bound to c-kit on Mo7e and efficiently transduced that cell line. Similarly, GALV or RD114 pseudotyped retroviral vector will normally not infect any mouse cells including mouse spermatozoa. However, if the envelope is altered by the inclusion of mouse membrane-bound SCF (the ligand for c-kit), binding of the pseudotyped vector using GALV or RD 114 concurrently with SCF may permit specific transduction of spermatozoa in vivo.

Preferably, in the method of the first aspect of the invention, the pseudotyped retroviral vector is a lentiviral vector. For example, the pseudotyped HIV-derived lentiviral vector as described by Naldini et al (1996) Science 272:263-67, or as used in a modified form in WO 00/29601 , is suitable.

The method of the first aspect of the invention is suitable for application to a variety of vertebrate animals. Thus, in accordance with the invention, a nucleic acid molecule of interest may be imparted to animals, including mammals, such as humans, non-human primates, for example simians, marmosets, domestic agricultural animals such as sheep, cows, pigs, horses, particularly race horses, marine mammals, feral animals, rodents such as mice and rats, and the like. Pigs and other large mammals are of particular interest, as their organs may be made suitable for xenotransplantion by the method of the invention. Other animals include birds such as chickens, turkeys, ducks, ostriches, geese, rare and ornamental birds, and the like. Also of interest are fish, including salmon, cod or zebrafish. Of particular interest are endangered species of wild animal, such rhinoceros, tigers, cheetahs, certain species of condor, and the like.

It will be appreciated that the selection of the nucleic acid molecule of interest will depend upon which species the method is to be applied to, and for what purpose.

In a preferred embodiment, the nucleic acid molecule of interest encodes a protein, preferably a therapeutic protein. By "therapeutic protein" we include the meaning that the protein is of therapeutic value to the transgenic vertebrate in which it is expressed. In addition to, or instead of the protein being of therapeutic benefit to the transgenic vertebrate in which it is expressed, it may be of therapeutic benefit in human or veterinary medicine or well being after recovery from the transgenic vertebrate in which it is expressed. For example, the therapeutic protein may be produced in the milk or other secretions of cows or

other animals. It may be, for example, a factor that enhances blood clotting in haemophiliacs, or a hormonal agent such as insulin or another peptide hormone. As a further alternative, the expression of the therapeutic protein may allow the transgenic vertebrate to produce a pharmaceutical of use in human or veterinary medicine, where the pharmaceutical is not itself the therapeutic protein. For example, in this context, the therapeutic protein may be a metabolic enzyme that allows for the production of a useful metabolite.

In another preferred embodiment, the nucleic acid molecule of interest encodes a ribonucleic acid molecule, preferably a ribozyme or antisense RNA.

A ribozyme is capable of cleaving targeted RNA or DNA. For example, the RNA or DNA to be cleaved may encode an undesirable protein. Suitable ribozymes are described in Cech and Herschlag Site-specific cleavage of single stranded DNA (US 5,180,818); Altman et al Cleavage of targeted RNA by RNAse P (US 5,168,053); Cech et al RNA ribozyme restriction endoribonucleases and methods (US 5,116,742); Been et al RNA ribozyme polymerases, dephosphorylases, restriction endonucleases and methods (US 5,093,246); and Been et al πRNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods; cleaves single-stranded RNA at specific site by transesterificatiopUS 4,987,071), all incorporated herein by reference.

By "antisense RNA" we mean an RNA molecule which hybridises to, and interferes with the expression from a mRNA molecule encoding a protein or to another RNA molecule within the cell such as pre-mRNA or tRNA or rRNA, or hybridises to, and interferes with the expression of a gene.

Conveniently, a gene expressing an antisense RNA may be constructed by inserting a sequence encoding a protein adjacent to a promoter in the appropriate orientation such that the antisense RNA is complementary to mRNA. Suitably, the antisense RNA blocks expression of undesirable polypeptides.

It will be appreciated that it may be sufficient to reduce expression of the undesirable polypeptide rather than abolish the expression.

It will be further appreciated that DNA sequences suitable for expressing as antisense RNA may be readily derived from publicly accessible databases such as GenBank and EMBL

In a preferred embodiment, the nucleic acid molecule of interest is capable of correcting or ameliorating a genetic disease. For example, where the genetic disease is caused by a lack of expression, or insufficient expression of a particular gene, the nucleic acid molecule may encode that particular gene. Where the genetic disease is caused by the inappropriate expression of a gene, particularly a mutated gene, the nucleic acid molecule of interest may reduce or abolish the expression of that gene.

There are several thousand inherited genetic diseases of mammals, including humans, that are caused by defective genes. Examples of such genetic diseases include cystic fibrosis, where there is known to be a mutation in the CFTR gene; Duchenne muscular dystrophy, where there is known to be a mutation in the dystrophin gene; sickle cell disease, where there is known to be a mutation in the HbA gene. Many types of cancer are caused by defective genes, especially protooncogenes, and tumour-suppressor genes that have undergone mutation.

Thus, it is preferred that the nucleic acid molecule of interest encodes a functionai proto-oncogene, or tumour-suppressor gene to replace the function of the defective proto-oncogene or tumour-suppressor gene.

Examples of proto-oncogenes are ras, src, bcl and so on; examples of tumour- suppressor genes are p53 and Rb.

Typically, a therapeutic protein or polypeptide will be one that a patient is unable to synthesise in his or her body or does not synthesise in the usual amount. An example of this is the use of gene therapy to treat adenosine deaminase dependent severe combined immunodeficiency (ADA - SCID). However, the concepts described herein are applicable to situations in which the nucleic acid molecule of interest encodes a protein or polypeptide that binds a substance that is overexpressed in a patient's body, e.g. causing some harmful physiological effect, or a protein or polypeptide that can bind to a polypeptide that is produced in a patient's body in an inactive form to activate it or in an active form to

inactivate it. Preferably, the use of the present invention in these applications has the advantage that the therapy provided by transfecting the stem cells is long lasting or permanent, thereby helping to avoid the need for frequently repeated treatment.

Diseases that might be treated using the methods described herein include all forms of chronic granulomatous disease (CGD), all forms of severe combined immunodeficiency (SCID), hyper gamma globulinaemia syndrome (hyper IgM), Wiskott-Aldrich Disease (WAS), thallassaemia, sickle-cell anaemia, other anaemias due to deficiencies of red blood cell proteins, neutrophil defects due to failure to synthesise granule components, e.g. myeloperoxidase deficiency, haemophilia and other clotting disorders such as complement deficiencies, lysomal storage disorders, such as Gaucher's disease, Hurler's disease, and mucopolysaccharidosis, leukocyte adhesion deficiency (LAD), bare lymphocyte syndrome, cancer and AIDS.

A specific reproductive application of the present method is to the treatment of male animals, particularly male humans, with subfertility or sterility due, for example, to a motility disorder of the spermatozoa with a genetic basis.

The present method is further applicable to the generation of transgenic animals of a suitable anatomical and physiological phenotype for xenograft transplantation, particularly to a human recipient. Transgenic technology permits the generation of animals which are immune-compatible with a human recipient. Appropriate organs, for example, may be removed from such animals to allow the transplantation of, for example, the heart, lung and kidney. Organs may alternatively be for extracorporal use, for example pig livers may be used in extracorporal blood purification for human patients, for example they may be dissociated and used in bioreactors for blood purification.

The present method is further applicable to improving breeding strategies for vertebrates, particularly livestock, by reducing susceptibility of the vertebrate to disease. This may be achieved by abolishing or reducing expression of such receptors on cells that are used by microorgansisms such as viruses or bacteria to infect the vertebrate. Modified receptors may be introduced by the method of the invention, to permit physiological functions of the receptor to be performed

within the vertebrate (Gordon JW and Ruddle FH (1983) gene transfer to mouse embryos: Production of transgenic mice by pronuclear injection. Methods Enzymol. 101:411-433. Any gene may be introduced using this method.

A second aspect of the invention provides a method of obtaining a zygote comprising a nucleic acid molecule of interest which method comprises fertilizing a female germ cell with a transgenic mature spermatozoan obtainable by a method of the earlier aspects of the invention.

To obtain a zygote, it is usually necessary to use a female germ cell of the same species as that of the transgenic spermatozoan. However, it may be possible to use gametes of related but different species. For example, a zygote may be formed using a horse and a donkey gamete.

In a preferred embodiment, fertilization is effected in vitro. Suitable methods include in vitro fertilization, cloning and embryo transfer, intracytoplasmic spermatozoal microinjection, cloning and embryo splitting, and the like.

Preferably, where fertilization is effected in vitro, the method of the second aspect of the invention further comprises cryopreserving the zygote. The cryopreserved zygote may be used at a remote location to facilitate the establishment of various valued livestock or birds. This approach is also applicable to the preservation of endangered species.

Alternatively, the fertilization may be effected in vivo. Transgenic sperm obtainable by the method of the first aspect of the invention may be introduced into a suitable female by artificial insemination. By "suitable female" we mean a female in which a zygote may be produced from the fusion of the transgenic spermatozoan with a gamete of the female. Usually, the female is of the same species as the male, or a closely related species.

Where the transgenic spermatozoan obtainable by the first aspect of the invention is present in the male vertebrate following administration of a pseudotyped viral vector to an epididymis or vas deferens, the method of the second aspect of the invention may be accomplished by natural mating of the recipient vertebrate and a suitable female vertebrate to produce a zygote. This

embodiment is particularly suitable for species of vertebrate for which in vitro methods of fertilization and assisted reproduction means are poorly developed.

A third aspect of the invention provides a method of obtaining a transgenic vertebrate comprising a nucleic acid molecule of interest, which method includes a step of allowing the zygote obtained according to the second aspect of the invention to develop into a transgenic vertebrate.

Accordingly, the method of the third aspect of the invention is as follows:

i) Firstly, a transgenic spermatozoan is obtained by incorporating a nucleic acid molecule of interest into a mature spermatozoan. This step comprises exposing a mature spermatozoan of a vertebrate to a pseudotyped viral vector comprising the nucleic acid molecule of interest under conditions which allow for the introduction of said pseudotyped viral vector into the mature spermatozoan.

ii) Secondly, a transgenic zygote comprising the nucleic acid molecule of interest is obtained by fertilizing a female germ cell with the transgenic spermatozoan obtained in step (i).

iii) Thirdly, a transgenic vertebrate comprising the nucleic acid molecule of interest is obtained by allowing the transgenic zygote to develop into a transgenic vertebrate.

Where the zygote of the second aspect of the invention is obtained by natural mating or artificial insemination, the zygote may be allowed to develop into a transgenic vertebrate within the natural mother. Alternatively, an embryo arising from the zygote in the natural mother may be transferred to a surrogate mother. This procedure is well known for rodents, especially mice (B Hogan, R Beddington, F Costantini, E Lacy, 1994, Manipulating the Mouse Embryo: A Laboratory Manual Cold Spring Harbor Laboratory Press, New York). Where the zygote is obtained in vitro, the zygote, or an early embryo which has developed from the zygote, may be introduced into a suitable surrogate mother for development in utero, by methods known in the art (B Hogan et a/, supra). The nucleic acid molecule of interest is maintained in the cells of the transgenic

vertebrate. It is preferred that the nucleic acid molecule of interest is maintained in the germ line of the transgenic vertebrate.

A fourth aspect of the invention provides a method of obtaining a transgenic vertebrate progeny comprising a nucleic acid molecule of interest comprising breeding the transgenic vertebrate obtained by the method of the third aspect of the invention, or a transgenic descendant thereof, to obtain a transgenic vertebrate progeny. The nucleic acid molecule of interest is maintained in the cells of the transgenic vertebrate progeny. It is preferred that the nucleic acid molecule of interest is maintained in the germ line of the transgenic vertebrate progeny.

It will be readily understood that breeding may be brought about by natural mating, or by in vitro or in vivo artificial means, as discussed above in relation to the second and third aspects of the invention.

A fifth aspect of the invention provides a method of providing a sample containing a transgenic spermatozoan comprising obtaining a sample containing the transgenic spermatozoan obtained by the method of the first aspect of the invention.

Preferably, the method further comprises the step of enriching the sample for transgenic spermatozoa. By "enriching" we mean that the proportion of cells in the sample that are transgenic spermatozoa is substantially greater after the enrichment step has been employed than before the enrichment step has been employed. Typically, the sample may contain transgenic spermatozoa and non- transgenic spermatozoa. Where the transgenic spermatozoa express a selectable marker, they may be enriched by a method that can distinguish cells expressing the selectable marker and cells which do not express the selectable marker. Where the selectable marker is a fluorescent marker, the transgenic spermatozoa may be enriched by processing the sample through a fluorescence activated cell sorter (FACS). Where the selectable marker is a membrane- anchored protein, the transgenic spermatozoa may be enriched using suitable binding reagents and capture on paramagnetic beads.

A sixth aspect of the invention provides a kit of parts comprising:

a pseudotyped viral vector suitable for incorporating a nucleic acid molecule comprising a selectable marker into a mature spermatozoan according to the method of the first aspect of the invention wherein the selectable marker is a protein which confers resistance to a toxin; and

the toxin.

The toxin may be used to kill spermatozoa and other cells that do not express the selectable marker. Accordingly, the kit provides means for obtaining transgenic spermatozoa according to the method of the first aspect of the invention, and means for selecting transgenic spermatozoa from spermatozoa that have not been made transgenic by the method of the invention.

Suitable toxins include geniticin (G418), puromycin, blasticidin, hygromycin, or histidinol.

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The invention will now be described in more detail, for the purposes of illustration only, in the following Examples and Figures.

Figure 1 - Enhanced transduction of M07e cells by Lentiviruses with surface SCF

Human MO7e cells with ckit receptors were infected with a Pgk-GFP lentiviral vector pseudotyped with either RD114 (feline specific), GALV (nonhuman primate specific) or VSV-G (universal). The % of GFP + cells infected by these viral vectors are shown as Dark-grey (originally blue) bars. Mid-grey bars (originally purple) show the percentage of GFP+ cells infected with the same viral envelopes but with hSCF. The hSCF on the envelope significantly enhances the infection rate of the lentiviral vectors even in the case of VSV-G that has a universal envelope.

Figure 2 - Western analysis of targeted viral envelopes showing the presence of pSCF

Various viral envelopes (GaIv, RD114 and VSV) were engineered so that hSCF was deployed on their surface. These targeted envelopes were purified and used for Western blot analysis. A primary antibody to hSCF was used to identify hSCF proteins and a peroxidase-labelled second antibody was used for visualization. Natural envelopes that had not been engineered with SCF were used as controls. Only the engineered envelopes showed the presence of SCF.

Figure 3 - Pig Spermatozoa showing c-kit on their perinuclear cell surface

Pig spermatozoa were stained with FITC-conjugated anti-ckit antibody (light grey - originally green) and DAPI (dark grey - originally blue) nuclear stained and were imaged on a Leica light microscope 6Ox (a). The same field of spermatozoa were imaged with phase contrast at the same magnification (b).

Figure 4 - 1-DE gel of newly synthesized pig spermatozoa proteins

Pig spermatozoa were incubated with S35 methionine. After incubation proteins were extracted and run on a 1-DE acrylimide gel. The two lanes represent duplicate samples. The gel was dried and the radioactive protein bands were imaged using a phosphoimager after a one week exposure.

Figure 5 - Western Blot of Phosphorylated Pig Spermatozoa Proteins

Pig spermatozoa were incubated with pSCF (c-kit ligand) for 5, 30 and 60 minutes and then run on 1-D acrylimide gel. The gel was blotted and the blot was probed with a radioactively labelled anti-phosphotyrosine antibody (PY99 Santa Cruz). The 60m membrane only column shows phoshporylated proteins that are specifically on the cell membrane.

Figure 6 - FACS Analysis of GFP Expression of Lentiviral infected Pig Spermatozoa

Pig spermatozoa were infected with a pseudotyped lentiviral vector VSVG-H2B- GFP. a. Shows the side scatter of the spermatozoa population (P1, predominantly light grey (originally orange)), b. shows the viable population of spermatozoa that were 7AAD negative (P3, light grey) and c. shows the viable GFP positive cells (GFP+, dark grey (originally green)).

Figure 7 - Presence of WPRE vector sequences DNA from infected spermatozoa

DNA was extracted from mouse and pig spermatozoa that had been infected with the lentiviral vector. The vector has woodchuck hepatitis post-regulatory sequences (WPRE) that enhance the expression of the transgene (Oh et al., 2007). Here we use primers to these sequences to detect the viral vectors in transduced spermatozoa DNA. Five out of seven transduced samples of pig spermatozoa DNA and 9 out of nine mouse samples had WPRE sequences. This indicates successful infection of mature spermatozoa by lentiviral vectors.

Figure 8 - Confocal images of spermatozoa

Confocal images were taken with a Zeiss 510inv confocal microscope with a 488mm laser line, (a) Control Spermatazoa, (b) Lentiviral infected spermatozoa under white light shows a green colouration even under an external light source , (c) Lentiviral infected spermatozoa under white light shows green fluorescence making the spermatozoa visible in the absence of other light sources

Figure 9 - A cDNA structure of soluble and membrane-bound forms of SCF.

cDNA reverse-transcribed from sSCF mRNA, which contains exon 6, generates a 935bp band by PCR using MF and MR primers. cDNA reverse- transcribed from mb-SCF, which lacks exon 6, generates a 851 bp band by PCR using the same primers. B PCR amplification of SCF or control sequences from cDNA reverse-transcribed from murine testicular tissue.

Lanes number 2 shows 935 bp and 851 bp bands corresponding to sSCF

and mb-SCF respectively; lane 3 is a positive control for the RT-PCR using PCR primers for GAPDH 5' and 3' yielding band of 350 bp as predicted

Figure 10 - Schematic diagrams of sperm maturation processes A transduction of spermatozoa in vitro; and B transduction of spermatozoa in vivo.

B includes a diagram showing the location of the epididymis and the vas deferens in relation to the testis.

Figure 11 - LAM-PCR of Transduced 293 Cells and Pig Sperm

Products are amplified viral-host junctions, extending to the nearest HAeIII site in the host genomic DNA. Bands in mock-infected cells derive from contaminating residual vector DNA. This artefact is out-competed if any other template is present.

Example 1 - Production of pseυdotyped lentiviral vectors

Chandrashekran et al., 2004 Blood 104(9):2697-703 (also see WO 2004/007735 (Imperial College Innovations Limited)) have developed a novel method for pseudotyping viral vectors with specific ligands on the envelope surface. Viral envelopes such as GALV and RD114 are specific for non-human primates and similarly, Eco are specific for mouse cells and will not infect cells from other species.

In contrast, pseudotyping viruses with the universal VSV-G envelopes, enables viruses to have a wide infection range which encompases nearly all cell types from numerous different species. Targeting viruses can be generated by combining a specific ligand molecule with a suitable envelope so that the virus can only bind and infect those cells that have a receptor for the ligand on their cell surface.

For instance a virus with an Eco mouse specific envelope with human SCF on it will only infect human cells that are c-kit positive (Figure 1). A human cell line MO7e that has a large number of ckit receptors on the cell surface was used to test the specificity of the targeted lentivirus. The MO7e cell line used herein was obtained from Dr Junia MeIo of Imperial College London.

A lentiviral vector consisted of GFP driven by the ubiquitous pgk promoter as pseudotyped with various envelopes with different tropisms: RD114 (feline specific), GALV (nonhuman primate specific) and VSV-G (universal). The same envelopes were engineered to have human membrane bound SCF attached to their surface.

These pseudotyped viral vectors with or without SCF (c-kit ligand) were used to infect MO7e cells and the number of cells expressing the reporter gene GFP was measured by FACS analysis (Figure 1 ).

These results show that the presence of SCF on the viral envelope was increased the infection in the case of VSV-G, RD114 and GALV pseudotyped viruses as expected, but interestingly, SCF was essential for the infection of viral vectors pseudotyped with the ecotropic envelope.

Example 2 - Demonstration of SCF on the viral envelope

Figure 1 shows that viral vectors with SCF on the envelope surface had a greater infectivity rate of ckit positive cells than pseudotyped viral vectors without SCF.

To demonstrate that these viral envelopes had SCF on their surface,, protein was extracted from the viral envelopes and run on a polyacrylamide gel for a Western blot analysis. The gel was blotted and the membrane was incubated with antibodies to hSCF (1 :500, R and D Systems) and probed with a radioactively labelled goat antihuman second antibody (1 :200, Dako). Viral envelopes that had been engineered to have SCF on their surface showed a radioactively labelled band while those that did not have SCF showed no band (Figure 2).

Example 3 - Demonstration of c-kit on the surface of pig spermatozoa

Pig spermatozoa have c-kit on their cell surface thus making them ideal targets for pSCF-targeted viruses. This was confirmed in using a FITC-labelled ant-c-kit antibody (Figure 3).

c-kit receptors were detected on the perinuclear cell surface of mature pig spermatozoa (green) while the nucleus was stained with DAPI (blue) (Figure 3a). Figure 3b shows the same field under phase contrast.

Example 4 - Demonstration of active protein synthesis by pig spermatozoa

1 ml of commercially available pig spermatozoa was washed twice in dBPS and resuspended in 10OuI PBS containing calcium, magnesium, pyruvate and glucose. 100ul of Dulbecco's medium (lacking methionine) supplemented with 10% dialysed FCS, penicillin, streptomycin and l-glutamine, further supplemented with trans S35 labelled methionine (30 uCi/ml) (NEN), was then added to the sperm mixture and incubated for 4 hours at 37 C.

Spermatozoa were than washed twice in cold PBS and lysed in 1X RIPA buffer (containing protease inhibitors). Protein lysate were resolved on 10% polyacrylamide gel, dried down and exposed to an x-ray film for 10 days. The film was than developed using an automated film development apparatus.

The protein bands shown on the film represent newly synthesised proteins demonstrating active protein synthesis.

Hence, we show that ejaculated pig spermatozoa are able to synthesize proteins de novo (Figure 4) and that the common perception that protein synthesis is shut down in mature free-swimming spermatozoa is false.

Example 5 - Demonstration of active phosphorylation of pig spermatozoa proteins

Our previous studies showed that proteins are actively synthesized in mature pig spermatozoa (Figure 4). Here we show that some proteins are also newly phoshorylated in mature pig spermatozoa (Figure 5). Some of these proteins are found in the spermatozoa membrane (Figure 5, membrane only), c-kit is a membrane protein that requires phosphorylation for cell signalling and activation of downstream effector functions. In this assay all phoshorylated membrane proteins were identified which should include c-kit.

1 ml of commercially obtained pig spermatozoa were washed twice in PBS and resuspended in 10OuI of PBS. Either 10ng/m! recombinant SCF or 10ul of viral supernatant (lentiviral vectors pseudotyped with various envelopes with or without SCF) was added to the mixture for 5 minutes and 30 minutes.

Spermatozoa were than washed twice in cold PBS and lysed with IxRIPA buffer (50OuI). Protein samples were electrophoresed on 4-12% gradient polyacrylamide gel, transferred onto a nylon membrane, probed with the anti phosphor tyrosine antibody and detected using a HRP secondary conjugated antibody (Routine Western Blotting)

This antibody identifies the proteins that have undergone phophorylation as a result of being stimulated by pSCF. Pig SCF is a ligand for c-kit and therefore it is likely that these phoshorylated proteins are both c-kit itself and the downstream proteins from c-kit signalling. It is clear from these studies that effector functions, which require phosphorylation, are active in mature spermatozoa

Example 6 - Pig and mouse spermatozoa can be infected by lentiviruses and express the Pgk/GFP transgene

The efficiency of infection of mature pig spermatozoa is dependent on the viral vector and its pseudotype as well as the temperature of infection. One ml aliquots of mature (ejaculated) pig spermatozoa (PIC Ltdl) were centrifuged at 600 rpm for 3m and resuspended in 1 ml of pig sperm medium. 100μl of different viral vectors containing (1x10 ⁸ viral particles) with various pseudotyped lentiviral vectors was added to the mature pig spermatozoa aliquot.

Some of the pseudotyped viruses additionally, had pig SCF on the envelope and were shown to target c-kit positive cells such as mature spermatozoa. The mature spermatozoa were incubated at different temperatures for different time periods. After incubation, GFP expression in transgenic spermatozoa was quantified using FACS analysis Figure 6.

The viral vector containing the GFP gene under the control of the Pgk promoter with the H2B for nuclear targeting of GFP was found to have the highest level of expression as detected by FACS (Figure 6). The Pgk promoter is used as a ubiquitous promoter in many instances both in vivo and in vitro. Clearly in these experiments Pgk is able to drive the expression of GFP in transgenic spermatozoa.

Example 7 - Optimizing Conditions for Infection of Mature Pig Spermatozoa with Lentiviruses

We tested various conditions and viral vectors in order to maximize the rate of infection of the mature spermatozoa. We compared the infectivity of pSCF pseudotyped viral vectors with those that had the wild type VSV-G envelope. In addition, one set of vectors had the H2B sequence, which targets the GFP to histones. We also tested various times and temperatures of infection (Table 1 ).

Table 1: Conditions for Optimal Infection of Mature Pig Spermatozoa.

Table 1 shows FACS analysis of GFP expression in transgenic spermatozoa after lentiviral infection. Ejaculated boar semen from PIC Incorporated was divided into 1 ml aliquots, centrifuged at 600 rpm for 3 minutes and resuspended in 1 ml sperm medium. A different viral vector was added to each aliquot and incubated for various times at different temperatures. The samples were then analyzed by FACS to quantify the number of spermatozoa expressing GFP. The viral vector Pgk-H2B- GFP pseudotyped with pSCF targeted VSVG showed the highest level of expression (18.2%)

The optimal temperature for long-term survival of ejaculated pig sperm is 18 ⁰ C although mature spermatozoa in vivo are usually at around 32 ⁰ C. Lentiviral vectors also operate well at 32 ⁰ C although many researchers use 37 ⁰ C for lentiviral infection of cells in vitro. This experiment showed the highest rates of infection (18.2%) were obtained when the pSCF-VSV-G-Pgk-H2B-eGFP lentivira! vector was allowed to incubate with pig spermatozoa for 18 hrs at 32 ⁰ C. In experiment using the same conditions 38% of the spermatozoa were infected and expressed GFP.

Example 8 - Detection of the lentiviral vector WPRE sequences in infected pig and mouse spermatozoa

The pig and mouse spermatozoa that had been infected with the lentiviral vectors were analyzed for the presence of the transgene. DNA was extracted from the infected spermatozoa and subjected to PCR using primers for the WPRE sequences (Figure 7).

Extraction of DNA from spermatozoa:

Extraction of DNA from spermatozoa was carried out using a commercially available kit (Qiagen, DNeasy Tissue kit) following slightly modified protocol provided by the manufacturer. The sperm was preincubated with a modified lysis buffer (X2) containing 2OmM Tris.CI, pH8.0, 2OmM EDTA, 2OmM NaCI, 8OmM

DTT, 4% SDS and 250ug Proteinase K. The mixture was incubated at 55 ^° C for 1 hour and samples agitated from time to time. Following this incubation, the standard routine protocol determine by the manufacturer was carried out and

DNA eluted into 35ul volume of Tris buffer.

PCR for the detection of WPRE and eGFP gene sequences encoded by the lentiviral vector.

Fifty nanograms of the DNA extracted as above were subjected to PCR (35 cycles with annealing temperature of 55 ¹ C) analysis. The following primers were utilised

Example 9 - Infection of mature Mouse Spermatozoa with Lentiviral Vectors

Mature mouse spermatozoa were isolated from the vas deferens and the epididymus by puncturing these tubes with a 3OG needle and allowing the spermatozoa to "swim up" for 15 m into 200μl of Human Tubule Formula (HTF) medium or dPBS containing Calcium, magnesium, pyruvate and glucose at 37 ⁰ C in 5% CO ₂ . (Hogan et at., 1994 Manipulating the Mouse Embryo. Cold Spring Harbor Press, 2 ^nd Edition). The vas deferens and the epididymus were removed and 100μl of the targeted viral vector, mSCF-VSVG-H2b-pgk-GFP containing 1x10 ⁸ particles was added to the spermatozoa for 3 hrs at 37 ⁰ C in 5% CO ₂ .. After the incubation the viable infected spermatozoa were analyzed for GFP expression using FACS. Confocal images of spermatozoa were taken with a Zeiss 510inv confocal microscope with a 488mm laser line (Figure 8).

Example 10- Integration of lentivectors into pig spermatozoa DNA

To establish if the lentiviral vector DNA had integrated into the pig sperm DNA, linear amplification mediated polymerase chain reaction (LAM-PCR) was employed. The method is based on the following published article (Harkey et a/. (2007). Multi-arm high-throughput integration site detection: limitations of LAM- PCR technology and optimization for clonal analysis. Stem Cells Dev. 2007 Jun;16(3):381-92).

At least 3 bona-fide integration sites could be detected 2 of which appear to be unique to the pig genome (with no known homology to any other genomic database). As the pig genome is in-complete, these sequences remain to be determined. The other integration site maps imperfectly to Human Rhesus, Chimp homologies. Two other identifiable sequences may indicate episomal-circular forms of the vector DNA (see table 2 below).

Summary of LAM-PCR Data from Transduced Pig Sperm

Both transduced pig sperm DNA samples were amplified by LAM-PCR Product between 100-800 bp was gel purified and shot-gun cloned 10 random clones from each set sequenced and analyzed for LTR-Host junctions

Table 2

Example 11: Generation of transgenic animals

Transgenic animals would be made using transgenic spermatozoa prepared in vitro according to the preceding examples, for assisted reproduction techniques such as in vitro fertilisation or artificial insemination as illustrated in Figure 10A.

As an alternative to preparing transgenic spermatozoa in vitro, pseudotyped retroviral vectors carrying a gene of interest and optionally a selectable marker would be injected into the vas deferens or epididymis of a male animal. Transgenic offspring would be generated by natural mating or, transgenic spermatozoa would be collected for assisted reproduction techniques as illustrated in Figure 1OB.