TITLE OF THE INVENTION

LENTIVIRAL VECTORS COMPRISING MICRORNAS

INCORPORATION BY REFERENCE

Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; "application cited documents"), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference. More generally, documents or references are cited in this text, either in a Reference List before the claims, or in the text itself; and, each of these documents or references ("herein cited references"), as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to incorporation of tissue-specific microRNA (miRNA) target sites into lentiviral vectors for restricting transgene expression to target cells. In particular, the vectors of the invention can restrict transgene expression to motor neuron populations, while significantly reducing transgene expression in muscle cells, providing potentially therapeutic vectors for motor neuron diseases.

BACKGROUND OF THE INVENTION

MicroRNA (miRNA) is a form of single-stranded RNA, typically about 18-25 nucleotides in length, which is expressed in mammalian cells. miRNAs do not themselves code for the production of a protein. Instead, miRNAs regulate expression of endogenous target genes.

The DNA that encodes a miRNA includes the miRNA sequence and an approximate reverse complement. When transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base pair to form a double stranded structure known as a

stem-loop (Lagos-Quintana et al. 2003 RNA 9(2): 175-179). This stem-loop is trimmed in the nucleus and exported to the cytoplasm where the enzyme Dicer further cleaves the stem loop into a double stranded 18-25 nucleotide mature miRNA. Finally, the miRNA binds to a number of proteins collectively known as the RNA-Induced Silencing Complex (RISC) and the sense strand of the miRNA is degraded. The antisense strand is transported to the target mRNA by RISC. Binding of the miRNA antisense strand to the target mRNA occurs and facilitates post- transcriptional silencing of target gene expression. miRNA shares partial or complete sequence complementary to a part of one or more messenger RNAs (mRNAs). Animal miRNAs generally target a site in the 3' untranslated region (UTR), whereas plant miRNAs usually target coding regions of mRNAs. The annealing of the miRNA to the mRNA typically results in one of two actions: either the miRNA complex blocks the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded; or, miRNA facilitates the cleavage of the mRNA i.e., degradation. Degradation occurs when binding of the miRNA causes the formation of double-stranded RNA, which triggers the degradation of the mRNA transcript through a process similar to that mediated by short interfering RNA (siRNA).

Inhibition of protein translation is believed to occur through target methylation by miRNAs of genomic sites that correspond to targeted mRNAs. Here, miRNAs function in association with a complement of proteins collectively termed the microribonucleoprotein complex (miRNP). Studies indicate that mRNAs containing partial complementary miRNAs can be targeted for degradation in vivo, that miRNA-dependent regulation of mRNA stability may be more common than previously appreciated, and that this mode of gene regulation is likely to be an important part of the biological function of miRNAs.

Hundreds of distinct miRNA genes are known to exist and to be differentially expressed during development and across tissue types. Recent studies have suggested important regulatory roles for miRNAs in a broad range of biological processes including developmental timing, cellular differentiation, proliferation, apoptosis, oncogenesis, insulin secretion, and cholesterol biosynthesis. (See Bartel 2004 Cell 116:281-97; Ambros 2004 Nature 431 :350-55; Du et al. 2005 Development 132:4645-52; Chen 2005 N. Engl. J. Med. 353:1768-71; Krutzfeldt et al. 2005 Nature 438:685-89.) Only a few miRNA targets have been thoroughly analyzed experimentally; however, molecular analysis has shown that miRNAs have distinct expression profiles in

different tissues. While the targets of many miRNAs have not been identified, computational methods have been used to analyze the expression of approximately 7,000 predicted human miRNA targets. The data suggest that miRNA expression broadly contributes to tissue specificity of mRNA expression in many human tissues. (See Sood et al. 2006 PNAS USA 103(8):2746-51.)

Further elucidation of this complex network of gene regulation will play a crucial role in designing gene therapies for numerous diseases, including motor neuron diseases, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA).

Amyotrophic lateral sclerosis (ALS), sometimes called Lou Gehrig's disease, is a rapidly progressive neurodegenerative disease. ALS, which is invariably fatal, attacks the neurons responsible for controlling voluntary muscles. Generally, both the upper motor neurons and the lower motor neurons degenerate or die, ceasing to send messages to muscles. Unable to function, the muscles gradually weaken, waste away, and twitch. Eventually the ability of the brain to start and control voluntary movement is lost. Individuals with ALS lose their strength and the ability to move their arms, legs, and body. When muscles in the diaphragm and chest wall fail, individuals lose the ability to breathe without ventilatory support. The disease does not affect a person's ability to see, smell, taste, hear, or recognize touch, and it does not usually impair a person's thinking or other cognitive abilities. However, several recent studies suggest that a small percentage of patients may experience problems with memory or decision-making, and there is growing evidence that some may even develop a form of dementia.

SMA is a genetic disease caused by progressive degeneration of motor neurons in the spinal cord. The disorder causes weakness and wasting of the voluntary muscles. Weakness is often more severe in the legs than in the arms. The childhood SMAs are all autosomal recessive diseases, and the gene for SMA has been identified.

There are many types of SMA. SMA type I, also called Werdnig-Hoffmann disease, is evident before birth or within the first few months of life. There may be a reduction in fetal movement in the final months of pregnancy. Symptoms include floppiness of the limbs and trunk, feeble movements of the arms and legs, swallowing and feeding difficulties, and impaired breathing. Affected children never sit or stand and usually die before the age of 2.

Symptoms of SMA type II usually begin between 3 and 15 months of age. Children may have respiratory problems, floppy limbs, decreased or absent deep tendon reflexes, and twitching

of arm, leg, or tongue muscles. These children may learn to sit but will never be able to stand or walk. Life expectancy varies. ,

Symptoms of SMA type III (Kugelberg-Welander disease) appear between 2 and 17 years of age, and include abnormal manner of walking; difficulty running, climbing steps, or rising from a chair; and slight tremor of the fingers.

Kennedy's syndrome or progressive spinobulbar muscular atrophy may occur between 15 and 60 years of age. Features of this type may include weakness of muscles in the tongue and face, difficulty swallowing, speech impairment, and excessive development of the mammary glands in males. The course of the disorder is usually slowly progressive. Kennedy's syndrome is an X-linked recessive disorder, which means that women carry the gene, but the disorder only occurs in men.

Congenital SMA with arthrogryposis (persistent contracture of joints with fixed abnormal posture of the limb) is a rare disorder. Manifestations include severe contractures, curvature of the spine, chest deformity, respiratory problems, an unusually small jaw, and drooping upper eyelids.

Primate and non-primate lentiviral vectors are particularly effective gene transfer tools for the mammalian nervous system. (See e.g., U.S. Patent Nos. 6,924,123, 6,312,682, 6,312,683; Mitrophanous et al. 1999 Gene Then 6:1808-18.) The natural envelope of the lentivirus is generally replaced in the vector system with a heterologous envelope (referred to as pseudotyping). Pseudotyping confers on the vector the ability to transfer genes to a broad range of different cell types, including mammalian and non-mammalian cells. A wide range of envelope glycoproteins are capable of pseudotyping lentiviral vectors, including those from rabies virus, Mokola virus, Molony murine leukemia virus (MLV 4070A), gibbon ape leukemia virus, murine leukemia virus 10Al and Ebola virus. (See e.g., Mochizuki et al. 1998 J. Virol. 72:8873-83; Reiser 2000 Gene Ther. 7:910-13; Stitz et al. 2000 Virology 273:16-20; Kobinger et al. 2001 Nat. Biotechnol. 19:225-30). After injection into the brain, lentiviral vectors pseudotyped with vesicular stomatitis virus (VSV-G) envelope transduce neurons with consequent anterograde transport of the expressed protein throughout the cell bodies and axons. (See Mazarakis et al. 2001 Human Molec. Genet. 10(19):2109-21.)

Retrograde axonal transport is believed to mediate the entry into the central nervous system (CNS) of a number of viruses such as polio, herpes and rabies. (See Ohka et al. 1998

Virology 250:67-75; Kristensson et al. 1982 /. Neurol. ScL 54:149-56; Charlton et al. 1994 Curr. Top. Microbiol. Immunol. 187:95-119.) Neurotrophic factors and molecules such as tetanus toxin can also be transported to the CNS after binding to specific receptors at nerve endings, with subsequent internalization and fast axoplasmic transport. (See Hirokawa 1997 Curr. Opin. Neurobiol. 7:605-14.) There have only been a few reports involving the delivery of foreign genes to the CNS via peripheral injection. One study involved injection of herpes simplex virus (HSV) vectors into the sciatic nerve and footpad of mice. (Dobson et al. 1990 Neuron 5:353-60.) Another study with a herpes viral vector encoding proproenkephalin reported gene transfer to dorsal root ganglia and antihyperalgesic effects after delivery to abraded skin of the dorsal hindpaw of mice. (Wilson et al. 1999 PNAS USA 96:3211-16.) Others have used adenovirus with injection in the tongue or skeletal muscle or direct injection into the sciatic nerve or gastrocnemius muscle has also resulted in gene expression. (Ghadge et al. 1995 Gene Ther. 2:132-37; Kuo et al. 1995 Brain Res. 705:31-38; Baumgartner et al. 1998 Exp. Neurol. 153:102-12; Haase et al. 1998 J. Neurol. Sci. 160(Suppl. l):S97-105; Glatzel et al. 2000 PNAS USA 97:442-47; Perrelet et al. 2000 Eur. J. Neurosci. 12:2059-67.) In addition, naked DNA injection into the sciatic nerve or gastrocnemius muscle has also resulted in gene expression. (Sahenk et al. 1993 Brain Res. 606:126-29.) However, most of these methods have proven very inefficient in achieving significant gene transfer or long-lasting therapeutic results.

On the other hand, gene transfer experiments to neural cells, both in vivo and in vitro, using minimal lentiviral vectors pseudotyped with rabies-G viral envelope glycoproteins, have demonstrated enhanced gene transfer to neurons due to retrograde axonal transport and transduction of distal neurons connected to the site of injection. (Mazarakis et al. 2001 Human Molec. Genet. 10(19):2109-21.) This includes, for example, pseudotyping EIAV vectors with Rabies-G envelope proteins, which allows for retrograde transport of the vector such that intramuscular delivery of the vector results in transduction of and transgene expression in motor neuron populations innervating the injected muscle. Such vectors are potential therapeutic agents for treating motor neuron diseases such as ALS and SMA. One potential concern with this approach is the background expression of the therapeutic transgene in non-target cells, namely muscle cells.

SUMMARY OF THE INVENTION

The present invention describes an approach for restricting expression of a transgene to a target cell population by silencing transgene expression in non-target cell types by using endogenous microRNA species. MicroRNA (rniRNA) induces sequence-specific post- transcriptional gene silencing in many organisms, either by inhibiting translation of messenger RNA (mRNA) or by causing degradation of the mRNA. The present invention provides polynucleotide constructs and compositions useful in miRNA-mediated suppression of transgene expression in non-target cells, namely muscle cells. It therefore provides methods for using miRNA to restrict expression of the transgene to certain target cell populations, namely motor neurons.

A recent study has utilized endogenous miRNAs to inhibit transgene expression from lentiviral vectors in hematopoietic lineages. (Brown et al. 2006 Nature Med. 12(5):585-91., also published in WO2007/000668) Multiple copies of a 23-bp sequence with perfect complementarity to a miRNA target sequence that is specifically expressed in cells of haematopoietic lineage were incorporated into the 3 '-untranslated region (UTR) of a green fluorescent protein (GFP) expression cassette within a lentiviral vector backbone. In vitro and in vivo analysis of GFP expression following vector transduction demonstrated that transgene expression was down-regulated in haematopoietic cells but was unaffected in other cell types.

Three miRNAs, miR-lb/d, miR-133, and miR-206, have been identified with tissue- biased expression profiles in muscle. In a preferred embodiment, the present invention utilizes these miRNAs in a lentiviral-mediated transgene delivery system by incorporating one or more copies of the miRNA target site into the lentiviral vector to increase specificity of transgene expression in motor neuron populations, while inhibiting expression in transduced muscle cells following intramuscular delivery.

Therefore, one aspect of this invention provides a vector pseudotyped with rabies G- protein, wherein the vector comprises a muscle cell-specific miRNA target sequence. In a preferred embodiment, the muscle cell-specific miRNA is operably linked to a nucleotide sequence of interest (NOI)

Another aspect of this invention provides a method for expressing a NOI in a target cell, comprising the steps of: injecting a vector comprising the NOI intramuscularly, wherein the vector further comprises a muscle cell-specific microRNA operably linked to the NOI,

transporting the vector from the muscle to the cell body of the target cell by retrograde transport, and expressing the NOI in the target cell, wherein the muscle cell-specific miRNA prevents, expression of the NOI in the muscle. Preferably the target cell is a neuron and the vector is pseudotyped with rabies G-protein for transporting the vector from the muscle to the cell body of the neuron by retrograde transport

Another aspect of the invention is the use of a vector comprising an NOI operably linked to a muscle cell-specific miRNA in the manufacture of a medicament for transducing a target site, wherein the vector travels to the target site by retrograde transport. Preferably the target site is a neuron and the vector is pseudotyped with rabies G-protein for transporting the vector from the muscle to the cell body of the neuron by retrograde transport.

In one embodiment, the target cell is in the spinal cord. In a preferred embodiment, the target cell is selected from the group consisting of sensory neurons motor neurons, interneurons, glial cells, astrocytes and oligodendrocytes.

In a preferred embodiment, the vector is a lentiviral vector, for example, an HIV-based lentiviral vector or an EIAV-based lentiviral vector. The vector can be a self-inactivating (SIN) vector.

The NOI can encode, for example, a siRNA or a gene product. In a preferred embodiment, the gene product is a protein. The protein can be a growth factor. In a preferred embodiment, the protein is selected from the group consisting of GDNF, IGF-I, VEGF, NT-3, CT-I, bcl-2, SMNl, SMN2, SODl and FVIII.

An additional aspect of the invention is a vector for tissue-specific expression of a nucleotide sequence of interest (NOI) in a target cell, wherein the vector comprises an NOI and at least one tissue-specific microRNA (miRNA) target sequence, wherein the miRNA target sequence restricts expression of the NOI to the target cell.

Another aspect of the invention is a method for tissue-specific expression of an NOI in a target cell comprising delivering a vector comprising an NOI and at least one tissue-specific

microRNA (miRNA) target sequence into the target cell, wherein expression of the NOI is restricted to the target cell.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic diagram of the invention.

Figure 2 shows the codon-optimized nucleotide sequence (SEQ ID NO: 9) encoding rabies G protein.

Figure 3 shows a schematic diagram of pONYKZmiRX.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, Ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice.; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

RETROVIRUSES

As it is well known in the art, a vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the present invention, and by way of example, some vectors used in recombinant DNA techniques allow entities, such as a heterologous segment of DNA (for example, a heterologous cDNA segment), to be transferred into a host cell for the purpose of replicating the vectors comprising a segment of DNA. Examples of vectors used in recombinant DNA techniques include, but are not limited to, plasmids, chromosomes, artificial chromosomes or viruses.

The term "expression vector" means a construct capable of in vivo or in vitro/ex vivo expression.

The retroviral vector employed in the aspects of the present invention may be derived from or may be derivable from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include: murine leukemia virus (MLV), human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV). A detailed list of retroviruses may be found in Coffin et al., 1997, "Retroviruses" Cold Spring Harbor Laboratory Press, Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763.

Retroviruses may be broadly divided into two categories: namely, "simple" and "complex." Retroviruses may even be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses. A review of these retroviruses is presented in Coffin et al. 1997 (supra).

In a typical vector for use in the present invention, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective. Portions of the viral genome may also be replaced by a library encoding candidate modulating moieties operably linked to a regulatory control region and a reporter moiety in the vector genome in order to generate a vector comprising candidate modulating moieties which is capable of transducing a target non-dividing host cell and/or integrating its genome into a host genome.

The basic structure of a retrovirus genome is a 5' long terminal repeat (LTR) and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome and gag, pol and env genes encoding the packaging components - the polypeptides encoded by these genes required for the assembly of viral particles. More complex retroviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.

In the provirus, the viral genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration, and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes. Encapsidation of the retroviral RNAs occurs by virtue of apsi sequence located at the 5' end of the viral genome.

The LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA and U5 is derived from the sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.

For the viral genome, the site of transcription initiation is at the boundary between U3 and R in one LTR and the site of poly (A) addition (termination) is at the boundary between R and U5 in the other LTR. U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins. Some retroviruses have any one or more of the following genes that code for proteins that are involved in the regulation of gene expression: tot, rev, tax and rex.

With regard to the structural genes gag, pol and env themselves, gag encodes the internal structural protein of the virus. Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome. The env gene encodes the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction leads ultimately to infection by fusion of the viral membrane with the cell membrane.

In a defective retroviral vector genome gag, pol and env may be absent or not functional. The R regions at both ends of the RNA are repeated sequences. U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome respectively.

Retroviruses may also contain "additional" genes which code for proteins other than gag, pol and env. Examples of additional genes include (in HIV), one or more of vif, vpr, vpx, vpu, tat, rev and nef. EIAV has (amongst others) the additional gene S2.

Proteins encoded by additional genes serve various functions, some of which may be duplicative of a function provided by a cellular protein. In EIAV, for example, tat acts as a transcriptional activator of the viral LTR (Derse and Newbold 1993 Virology 194:530-6; Maury et al. 1994 Virology 200:632-42). It binds to a stable, stem-loop RNA secondary structure referred to as TAR. Rev regulates and co-ordinates the expression of viral genes through rev-response elements (RRE) (Martarano et al. 1994 J. Virol. 68:3102-11). The mechanisms of action of these two proteins are thought to be broadly similar to the analogous mechanisms in the primate viruses. The function of S2 is unknown. In addition, an EIAV protein, Ttm, has been identified that is encoded by the first exon of tat spliced to the env coding sequence at the start of the transmembrane protein.

Preferably the viral vector capable of transducing a target non-dividing or slowly dividing cell is a lentiviral vector.

Lenti virus vectors are part of a larger group of retroviral vectors. A detailed list of lentiviruses may be found in Coffin et al. (1997, "Retroviruses" Cold Spring Harbor Laboratory Press, Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763). In brief, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to: the human immunodeficiency virus (HIV), the causative agent of human auto- immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non- primate lentiviral group includes the prototype "slow virus" visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types of retroviruses is that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et al. 1992 EMBO J. 11:3053-3058; Lewis and Emerman 19947. Virol 68:510-516). In contrast, other retroviruses - such as MLV - are unable to infect non-dividing or slowly dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.

A "non-primate" vector, as used herein in some aspects of the present invention, refers to a vector derived from a virus which does not primarily infect primates, especially humans. Thus, non-primate virus vectors include vectors which infect non-primate mammals, such as dogs, sheep and horses, reptiles, birds and insects.

A lentiviral or lentivirus vector, as used herein, is a vector which comprises at least one component part derivable from a lentivirus. Preferably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated. The term "derivable" is used in its normal sense as meaning the sequence need not necessarily be obtained from a retrovirus but instead could be derived therefrom. By way of example, the sequence may be prepared synthetically or by use of recombinant DNA techniques.

The non-primate lentivirus may be any member of the family of lentiviridae which does not naturally infect a primate and may include a feline immunodeficiency virus (FIV), a bovine immunodeficiency virus (BIV), a caprine arthritis encephalitis virus (CAEV), a Maedi visna virus (MVV) or an equine infectious anaemia virus (EIAV). Preferably the lentivirus is an EIAV. Equine infectious anaemia virus infects all equidae resulting in plasma viremia and thrombocytopenia (Clabough et al. 1991 J. Virol. 65:6242-51). Virus replication is thought to be controlled by the process of maturation of monocytes into macrophages.

In addition to protease, reverse transcriptase and integrase, non-primate lentiviruses contain a fourth pol gene product which codes for a dUTPase. This may play a role in the ability of these lentiviruses to infect certain non-dividing or slowly dividing cell types.

The viral RNA of this aspect of the invention is transcribed from a promoter, which may be of viral or non-viral origin, but which is capable of directing expression in a eukaryotic cell such as a mammalian cell. Optionally an enhancer is added, either upstream of the promoter or downstream. The RNA transcript is terminated at a polyadenylation site which may be the one provided in the lentiviral 3' LTR or a different polyadenylation signal.

Thus the present invention employs a DNA transcription unit comprising a promoter and optionally an enhancer capable of directing expression of a non-primate lentiviral vector genome.

Transcription units as described herein comprise regions of nucleic acid containing sequences capable of being transcribed. Thus, sequences encoding mRNA, tRNA and rRNA are included within this definition. The sequences may be in the sense or antisense orientation with respect to the promoter. Antisense constructs can be used to inhibit the expression of a gene in a cell according to well known techniques. Nucleic acids may be, for example, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogues thereof. Sequences encoding mRNA will optionally include some or all of 5' and/or 3' transcribed but untranslated flanking sequences

naturally, or otherwise, associated with the translated coding sequence. It may optionally further include the associated transcriptional control sequences normally associated with the transcribed sequences, for example transcriptional stop signals, polyadenylation sites and downstream enhancer elements. Nucleic acids may comprise cDNA or genomic DNA (which may contain introns).

Preferred vectors for use in accordance with one aspect of the present invention are recombinant non-primate lenti viral vectors.

The term "recombinant lentiviral vector" (RLV) refers to a vector with sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell includes reverse transcription and integration into the target cell genome. The RLV carries non-viral coding sequences which are to be delivered by the vector to the target cell. An RLV is incapable of independent replication to produce infectious retroviral particles within the final target cell. Usually the RLV lacks a functional gag-pol and/or env gene and/or other genes essential for replication. The vector of the present invention may be configured as a split-intron vector. A split intron vector is described in PCT patent application WO 99/15683.

Preferably the lentiviral vector of the present invention has a minimal viral genome. As used herein, the term "minimal viral genome" means that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in our WO 98/17815.

A minimal lentiviral genome for use in the present invention will therefore comprise (5')R-U5-one or more first nucleotide sequences-U3-R(3'). However, the plasmid vector used to produce the lentiviral genome within a host cell/packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell/packaging cell. These regulatory sequences may be the natural sequences associated with the transcribed retroviral sequence, i.e. the 5' U3 region, or they may be a heterologous promoter such as another viral promoter, for example the CMV promoter. Some lentiviral genomes require additional sequences for efficient virus production. For example, in the case of HIV, rev and RRE sequences are preferably included. However the requirement for rev and RRE may be reduced or eliminated by codon optimization. Further

details of this strategy can be found in our WO 01/79518. Alternative sequences which perform the same function as the rev/RRE system are also known. For example, a functional analogue of the rev/RRE system is found in the Mason Pfizer monkey virus. This is known as CTE and comprises an RRE-type sequence in the genome which is believed to interact with a factor in the infected cell. The cellular factor can be thought of as a rev analogue. Thus, CTE may be used as an alternative to the rev/RRE system. Any other functional equivalents which are known or become available may be relevant to the invention. For example, it is also known that the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-I . It is also known that Rev and Rex have similar effects to IRE-BP.

In our WO 99/32646 we give details of features which may advantageously be applied to the present invention. In particular, it will be appreciated that the non-primate lentivirus genome (1) preferably comprises a deleted gag gene wherein the deletion in gag removes one or more nucleotides downstream of about nucleotide 350 or 354 of the gag coding sequence; (2) preferably has one or more accessory genes absent from the non-primate lentivirus genome; (3) preferably lacks the tat gene but includes the leader sequence between the end of the 5' LTR and the ATG of gag; and (4) combinations of (1), (2) and (3). In a particularly preferred embodiment the lentiviral vector comprises all of features (1) and (2) and (3).

Expression may be controlled using control sequences, which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue-specific or stimuli-specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters.

Suitable promoting sequences are strong promoters including those derived from the genomes of viruses - such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), retrovirus and Simian Virus 40 (SV40) - or from heterologous mammalian promoters - such as the actin promoter or ribosomal protein promoter. Transcription of a gene may be increased further by inserting one or more enhancer sequences into the vector. Enhancers are relatively orientation- and position-independent, however, one may employ an enhancer from a eukaryotic cell virus - such as the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer. The enhancer

may be spliced into the vector at a position 5' or 3' to the promoter, but is preferably located at a site 5' from the promoter.

The promoter can additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions e.g. a Pribnow Box or a TATA box. The promoter may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of a nucleotide sequence. Suitable other sequences include the Shl-intron or an ADH intron. Other sequences may include inducible elements - such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present.

Disruption of the open reading frame of Tαt enhances the safety profile of the vectors with no detrimental effect on titer despite the fact that the first exon of Tαt is within the packaging signal. This disruption may be achieved by the insertion of a nucleotide within the initial codon of the Tαt open reading frame (plasmid nucleotides 1317-1319) in the vector genome. gttgaacCTG -> gttgaacCTCG (SEQ ID NOs: 1 and 2, respectively)

This was confirmed by sequencing and titering of the new genome which revealed no loss of titer resulting from this modification. Genomes without this modification express the amino-terminal portion (29 aa) of the viral protein Tat in producer cells.

The titer of vectors having a mutation in the major splice donor (SDl) is at least as high as those with a functional major splice donor. The disruption may be achieved by site-directed mutagenesis substituting nucleotide 1405 (T) for 'C thereby destroying the splice donor.

AGGT -> AGGC

The mutated splice donor is non-functional as tested by insertion of a functional splice acceptor downstream.

The presence of a sequence termed the central polypurine tract (cPPT) may improve the efficiency of gene delivery to non-dividing cells (see WO 00/31200). This ds-acting element is located, for example, in the EIAV polymerase coding region element. Preferably the genome of the vector system used in the present invention comprises a cPPT sequence.

Other types of elements can also be used to stimulate heterologous gene expression post- transcriptionally. For instance, woodchuck hepatitis virus (WHV) harbors a post-transcriptional regulatory element (PRE) (hereinafter referred to as WPRE; see U.S. Patent Nos. 6,136,597 and

6,287,814). The WPRE element enhances expression and as such is likely to be beneficial in attaining maximal levels of the NOL

In addition, or in the alternative, the viral genome may comprise a post-translational regulatory element and/or a translational enhancer.

MINIMAL SYSTEMS

Preferably the lentiviral vector of the present invention has a minimal viral genome. As used herein, the term "minimal viral genome" means that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in our WO 98/17815.

A minimal lentiviral genome for use in the present invention will therefore comprise (5') R - U5 - one or more first nucleotide sequences - U3-R (3'). However, the plasmid vector used to produce the lentiviral genome within a host cell/packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell/packaging cell. These regulatory sequences may be the natural sequences associated with the transcribed retroviral sequence, i.e. the 5' U3 region, or they may be a heterologous promoter such as another viral promoter, for example the CMV promoter.

It has been demonstrated that a primate lentivirus minimal system can be constructed which requires none of the HIV/SIV additional genes vif, vpr, vpx, vpu, tat, rev and nef for either vector production or for transduction of dividing and non-dividing cells. It has also been demonstrated that an EIAV minimal vector system can be constructed which does not require S2 for either vector production or for transduction of dividing and non-dividing cells.

The deletion of additional genes is highly advantageous. Firstly, it permits vectors to be produced without the genes associated with disease in lentiviral {e.g. HIV) infections. In particular, tat is associated with disease. Secondly, the deletion of additional genes permits the vector to package more heterologous DNA. Thirdly, genes whose function is unknown, such as S2, may be omitted, thus reducing the risk of causing undesired effects. Examples of minimal lentiviral vectors are disclosed in WO 99/32646 and in WO 98/17815.

Thus, preferably, the delivery system used in the invention is devoid of at least tat and S2 (if it is an EIAV vector system), and possibly also vif, vpr, vpx, vpu and nef. More preferably,

the systems of the present invention are also devoid of rev. Rev was previously thought to be essential in some retroviral genomes for efficient virus production. For example, in the case of HIV, it was thought that rev and RRE sequence should be included. However, it has been found that the requirement for rev and RRE can be reduced or eliminated by codon optimization (see below). As expression of the codon optimized gag-pol is Rev independent, RRE can be removed from the gag-pol expression cassette, thus removing any potential for recombination with any RRE contained on the vector genome.

Alternative sequences which perform the same function as the rev/RRE system are also known. For example, a functional analogue of the rev/RRE system is found in the Mason Pfizer monkey virus. This is known as CTE and comprises an RRE-type sequence in the genome which is believed to interact with a factor in the infected cell. The cellular factor can be thought of as a rev analogue. Thus, CTE may be used as an alternative to the rev/RRE system. Any other functional equivalents which are known or become available may be relevant to the invention. For example, it is also known that the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-I. It is also known that Rev and Rex have similar effects to IRE-BP.

In our WO 99/32646 we give details of features which may advantageously be applied to the present invention. In particular, it will be appreciated that the non-primate lentivirus genome (1) preferably comprises a deleted gag gene wherein the deletion in gag removes one or more nucleotides downstream of about nucleotide 350 or 354 of the gag coding sequence; (2) preferably has one or more accessory genes absent from the non-primate lentivirus genome; (3) preferably lacks the tat gene but includes the leader sequence between the end of the 5' LTR and the ATG of gag; and (4) combinations of (1), (2) and (3). In a particularly preferred embodiment the lentiviral vector comprises all of features (1) and (2) and (3).

CODON OPTIMIZATION

Codon optimization has previously been described in WO 99/41397. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately

choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available.

Many viruses, including HIV and other lentiviruses, use a large number of rare codons and by changing these to correspond to commonly used mammalian codons, increased expression of the packaging components in mammalian producer cells can be achieved. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms.

Codon optimization has a number of other advantages. By virtue of alterations in their sequences, the nucleotide sequences encoding the packaging components of the viral particles required for assembly of viral particles in the producer cells/packaging cells have RNA instability sequences (INS) eliminated from them. At the same time, the amino acid sequence coding sequence for the packaging components is retained so that the viral components encoded by the sequences remain the same, or at least sufficiently similar that the function of the packaging components is not compromised. Codon optimization also overcomes the Rev/RRE requirement for export, rendering optimized sequences Rev independent. Codon optimization also reduces homologous recombination between different constructs within the vector system (for example between the regions of overlap in the gag-pol and env open reading frames). The overall effect of codon optimization is therefore a notable increase in viral titer and improved safety.

In one embodiment only codons relating to INS are codon optimized. However, in a preferred and practical embodiment, the sequences are codon optimized in their entirety, with the exception of the sequence encompassing the frameshift site of gag-pol.

The gag-pol gene comprises two overlapping reading frames encoding the gag-pol proteins. The expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of ribosome "slippage" during translation. This slippage is thought to be caused at least in part by ribosome-stalling RNA secondary structures. Such secondary structures exist downstream of the frameshift site in the gag-pol gene. For HIV, the region of overlap extends from nucleotide 1222 downstream of the beginning of gag (wherein nucleotide 1 is the A of the gag ATG) to the end of gag (nt 1503). Consequently, a 281 bp fragment spanning the frameshift site and the overlapping region of the two reading frames is preferably not codon optimized. Retaining this fragment will enable more efficient expression of the gag-pol proteins.

For EIAV, the beginning of the overlap is at nt 1262 (where nucleotide 1 is the A of the gag ATG). The end of the overlap is at nt 1461. In order to ensure that the frameshift site and the gag-pol overlap are preserved, the wild type sequence has been retained from nt 1156 to 1465.

Derivations from optimal codon usage may be made, for example, in order to accommodate convenient restriction sites, and conservative amino acid changes may be introduced into the gag-pol proteins.

In a highly preferred embodiment, codon optimization was based on codons with poor codon usage in mammalian systems. The third and sometimes the second and third base may be changed.

Due to the degenerate nature of the Genetic Code, it will be appreciated that numerous gag-pol sequences can be achieved by a skilled worker. Also, there are many retroviral variants described which can be used as a starting point for generating a codon optimized gag-pol sequence. Lentiviral genomes can be quite variable. For example there are many quasi-species of HIV-I which are still functional. This is also the case for EIAV. These variants may be used to enhance particular parts of the transduction process. Examples of HIV-I variants may be found in the HIV databases maintained by Los Alamos National Laboratory. Details of EIAV clones may be found at the NCBI database maintained by the National Institutes of Health.

The strategy for codon optimized gag-pol sequences can be used in relation to any retrovirus. This would apply to all lentiviruses, including EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-I and HIV -2. In addition this method could be used to increase expression of genes from HTLV-I, HTLV-2, HFV, HSRV and human endogenous retroviruses (HERV), MLV and other retroviruses.

Codon optimization can render gag-pol expression Rev independent. In order to enable the use of anti-rev or RRE factors in the retroviral vector, however, it would be necessary to render the viral vector generation system totally Rev/RRE independent. Thus, the genome also needs to be modified. This is achieved by optimizing vector genome components. Advantageously, these modifications also lead to the production of a safer system absent of all additional proteins both in the producer and in the transduced cell.

As described above, the packaging components for a retroviral vector include expression products of gag, pol and env genes. In addition, efficient packaging depends on a short sequence

of 4 stem loops followed by a partial sequence from gag and env (the "packaging signal"). Thus, inclusion of a deleted gag sequence in the retroviral vector genome (in addition to the full gag sequence on the packaging construct) will optimize vector titer. To date efficient packaging has been reported to require from 255 to 360 nucleotides of gag in vectors that still retain env sequences, or about 40 nucleotides of gag in a particular combination of splice donor mutation, gag and env deletions. It has surprisingly been found that a deletion of all but the N-terminal 360 or so nucleotides in gag leads to an increase in vector titer. Thus, preferably, the retroviral vector genome includes a gag sequence which comprises one or more deletions, more preferably the gag sequence comprises about 360 nucleotides derivable from the N-terminus.

PSEUDOTYPING

In one preferred aspect, the retroviral vector of the present invention has been pseudotyped. In this regard, pseudotyping can confer one or more advantages. For example, with the lenti viral vectors, the env gene product of the HIV based vectors would restrict these vectors to infecting only cells that express a protein called CD4. But if the env gene in these vectors has been substituted with env sequences from other RNA viruses, then they may have a broader infectious spectrum (Verma and Somia 1997 Nature 389:239-242). By way of example, workers have pseudotyped an HIV based vector with the glycoprotein from VSV (Verma and Somia 1997 (ibid)).

In another alternative, the Env protein may be a modified Env protein such as a mutant or engineered Env protein. Modifications may be made or selected to introduce targeting ability or to reduce toxicity or for another purpose (Valsesia-Wittman et al. 1996 J. Virol. 70:2056-64; Nilson et al. 1996 Gene Therapy 3:280-6; Fielding et al. 1998 Blood 9:1802 and references cited therein).

The vector may be pseudotyped with any molecule of choice.

Rabies G

In the present invention the vector system may be pseudotyped with at least a part of a rabies G protein or a mutant, variant, homologue or fragment thereof.

Teachings on the rabies G protein, as well as mutants thereof, may be found in WO 99/61639 and well as Rose et al. 1982 J. Virol. 43: 361-364, Hanham et al. 1993 J. Virol. 67:530-542, Tuffereau et al.1998 J. Virol. 72:1085-1091, Kucera et al. 1985 /. Virol. 55:158- 162, Dietzschold et al. 1983 PNAS 80:70-74, Seif et al. 1985 J.Virol. 53:926-934, Coulon et al.

1998 /. Virol. 72:273-278, Tuffereau et al.1998 /. Virol. 72:1085-10910, Burger et al. 1991 J. Gen. Virol. 72:359-367, Gaudin et al. 1995 J Virol. 69:5528-5534, Benmansour et al. 1991 /. Virol. 65:4198-4203, Luo et al. 1998 Microbiol. Immunol. 42:187-193, Coll 1997 Arch. Virol. 142:2089-2097, Luo et al. 1997 Virus Res. 51:35-41, Luo et al. 1998 Microbiol. Immunol. 42:187-193, Coll 1995 Arch. Virol. 140:827-851, Tuchiya et al. 1992 Virus Res. 25:1-13, Morimoto et al. 1992 Virology 189:203-216, Gaudin et al. 1992 Virology 187:627-632, Whitt et al. 1991 Virology 185:681-688, Dietzschold et al. 1978 /. Gen. Virol. 40:131-139, Dietzschold et al. 1978 Dev. Biol. Stand. 40:45-55, Dietzschold et al. 1977 /. Virol. 23:286-293, and Otvos et al. 1994 Biochim. Biophys. Acta 1224:68-76. A rabies G protein is also described in EP 0445625.

The present invention provides a rabies G protein having the amino acid sequence shown in SEQ ID NO: 3. The present invention also provides a nucleotide sequence capable of encoding such a rabies G protein. Preferably the nucleotide sequence comprises the sequence shown in SEQ ID NO: 4.

These sequences differ from the GenBank sequence as shown below:

I Y T I L D K L (SEQ ID NO: 5)

GenBank Sequence ATT TAC ACG ATA CTA GAC AAG CTT (SEQ ID NO: 6)

I Y T I P D K L (SEQ ID NO: 7)

Present Invention ATT TAC ACG ATC CCA GAC AAG CTT (SEQ ID NO: 8)

In a preferred embodiment, the vector system of the present invention is or comprises at least a part of a rabies G protein having the amino acid sequence shown in SEQ ID NO: 3.

The use of rabies G protein provides vectors which, in vivo, preferentially transduce targeted cells which rabies virus preferentially infects. This includes in particular neuronal target cells in vivo. For a neuron-targeted vector, rabies G from a pathogenic strain of rabies such as ERA may be particularly effective. On the other hand rabies G protein confers a wider target cell range in vitro including nearly all mammalian and avian cell types tested (Seganti et al. 1990 Arch. Virol. 34:155-163; Fields et al. 1996 Fields Virology, Third Edition, vol.2, Lippincott- Raven Publishers, Philadelphia, New York).

The rabies G gene can also be codon optimized (see Figure 2; SEQ ID NO: 9).

The tropism of the pseudotyped vector particles may be modified by the use of a mutant rabies G which is modified in the extracellular domain. Rabies G protein has the advantage of

being mutatable to restrict target cell range. The uptake of rabies virus by target cells in vivo is thought to be mediated by the acetylcholine receptor (AchR) but there may be other receptors to which AchR binds in vivo (Hanham et al. 1993 /. Virol. 67:530-542; Tuffereau et al. 1998 J. Virol. 72:1085-1091). It is thought that multiple receptors are used in the nervous system for viral entry, including NCAM (Thoulouze et al. 1998 /. Virol. 72(9):7181-90) and p75 neurotrophin receptor (Tuffereau et al. 1998 EMBO J. 17(24):7250-9).

The effects of mutations in antigenic site III of the rabies G protein on virus tropism have been investigated, this region is not thought to be involved in the binding of the virus to the acetylcholine receptor (Kucera et al. 1985 /. Virol. 55:158-162; Dietzschold et al. 1983 PNAS 80:70-74; Seif et al. 1985 J. Virol. 53:926-934; Coulon et al.1998 J. Virol. 72:273-278; Tuffereau et al. 1998 /. Virol. 72:1085-10910). For example it has been reported that a mutation of the arginine at amino acid 333 in the mature protein to glutamine (i.e. ERAsm) can be used to restrict viral entry to olfactory and peripheral neurons in vivo while reducing propagation to the central nervous system. It has also been reported that these viruses were able to penetrate motor neurons and sensory neurons as efficiently as the wild type virus, yet transneuronal transfer did not occur (Coulon et al. 1989 J. Virol. 63:3550-3554). Viruses in which amino acid 330 has been mutated are further attenuated (i.e. ERAdm), and were reported as being unable to infect either motor neurons or sensory neurons after intra-muscular injection (Coulon et al.1998 J. Virol. 72:273-278).

Alternatively or additionally, rabies G proteins from laboratory passaged strains of rabies may be used. These can be screened for alterations in tropism. Such strains include the following:

Table 1.

By way of example, the ERA strain is a pathogenic strain of rabies and the rabies G protein from this strain can be used for transduction of neuronal cells. The sequence of rabies G from the ERA strains is in the GenBank database (Accession number J02293). This protein has a signal peptide of 19 amino acids and the mature protein begins at the lysine residue 20 amino acids from the translation initiation methionine. The HEP-Flury strain contains the mutation from arginine to glutamine at amino acid position 333 in the mature protein which correlates with reduced pathogenicity and which can be used to restrict the tropism of the viral envelope.

WO 99/61639 discloses the nucleic and amino acid sequences for a rabies virus strain ERA (GenBank locus RAVGPLS, Accession no. M38452).

In the present invention the vector system may be pseudotyped with at least part of a protein from the Challenge Virus Standard (CVS) strain of rabies virus, and in particular the CVS glycoprotein G, or a mutant, variant, homologue or fragment thereof. The cDNA for CVS rabies virus G is different in nucleotide sequence from ERA rabies virus G; teachings on CVS can be found in US Patent No. 5,348,741. ATCC deposit No. 40280, designated pKB3-JE-13, may conveniently be used in the present invention.

It will also be appreciated that CVS glycoproteins from laboratory passaged strains of CVS may be used. These can be screened for alterations in tropism.

It will further be appreciated that the instant invention encompasses vectors encoding equivalents of rabies G glycoprotein.

Accession information is provided merely as convenience to those of skill in the art, and are not an admission that deposits are required under 35 U.S.C. §112. The viral strains are incorporated herein by reference and are controlling in the event of any conflict with the description herein.

VSV-G

The envelope glycoprotein (G) of Vesicular stomatitis virus (VSV), a rhabdovirus, is another envelope protein that has been shown to be capable of pseudotyping certain retroviruses.

Its ability to pseudotype MoMLV-based retroviral vectors in the absence of any retroviral envelope proteins was first shown by Emi et al. (1991 J. Virol. 65:1202-1207). WO 94/294440 teaches that retroviral vectors may be successfully pseudotyped with VSV-G. These pseudotyped VSV-G vectors may be used to transduce a wide range of mammalian cells. Even

more recently, Abe et al. (1998 /. Virol. 72(8):6356-6361) teach that non-infectious retroviral particles can be made infectious by the addition of VSV-G.

Burns et al. (1993 PNAS USA 90:8033-7) successfully pseudotyped the retrovirus MLV with VSV-G and this resulted in a vector having an altered host range compared to MLV in its native form. VSV-G pseudotyped vectors have been shown to infect not only mammalian cells, but also cell lines derived from fish, reptiles and insects (Burns et al. 1993 (ibid)). They have also been shown to be more efficient than traditional amphotropic envelopes for a variety of cell lines (Yee et al. 1994 PNAS USA 91:9564-9568, Emi et al. 1991 J. Virol. 65:1202-1207). VSV-G protein can be used to pseudotype certain retroviruses because its cytoplasmic tail is capable of interacting with the retroviral cores.

The provision of a non-retroviral pseudotyping envelope such as VSV-G protein gives the advantage that vector particles can be concentrated to a high titre without loss of infectivity (Akkina et al. 1996 /. Virol. 70:2581-5). Retrovirus envelope proteins are apparently unable to withstand the shearing forces during ultracentrifugation, probably because they consist of two non-covalently linked subunits. The interaction between the subunits may be disrupted by the centrifugation. In comparison the VSV glycoprotein is composed of a single unit. VSV-G protein pseudotyping can therefore offer potential advantages.

WO 00/52188 describes the generation of pseudotyped retroviral vectors, from stable producer cell lines, having vesicular stomatitis virus-G protein (VSV-G) as the membrane- associated viral envelope protein, and provides a gene sequence for the VSV-G protein.

Ross River Virus

The Ross River viral envelope has been used to pseudotype a nonprimate lentiviral vector (FIV) and following systemic administration predominantly transduced the liver (Kang et al. 2002). Efficiency was reported to be 20-fold greater than obtained with VSV-G pseudotyped vector, and caused less cytotoxicity as measured by serum levels of liver enzymes suggestive of hepatotoxicity.

Ross River Virus (RRV) is an alphavirus spread by mosquitoes which is endemic and epidemic in tropical and temperate regions of Australia. Antibody rates in normal populations in the temperate coastal zone tend to be low (6% to 15%) although seroprevalence reaches 27 to 37% in the plains of the Murray Valley River system. In 1979 to 1980, RRV became epidemic

in the Pacific Islands. The disease is not contagious between humans and is never fatal, the first symptom being joint pain with fatigue and lethargy in about half of patients (Fields Virology).

Baculovirus GP64

The baculovirus GP64 protein has been shown to be an attractive alternative to VSV-G for viral vectors used in the large-scale production of high-titer virus required for clinical and commercial applications (Kumar et al. 2003 HMm. Gene Ther. 14(l):67-77). Compared with VSV-G, GP64 vectors have a similar broad tropism and similar native titers. Because GP64 expression does not kill cells, 293T-based cell lines constitutively expressing GP64 can be generated.

Alternative envelopes

Other envelopes which give reasonable titer when used to pseudotype EIAV include Mokola, Rabies, Ebola and LCMV (lymphocytic choriomeningitis virus). Following in utero injection in mice the VSV-G envelope was found to be more efficient at transducing hepatocytes than either Ebola or Mokola (Mackenzie et al. 2002). Intravenous infusion into mice of lentivirus pseudotyped with 4070A led to maximal gene expression in the liver (Peng et al. 2001).

NUCLEOTIDES OF INTEREST

In a broad aspect, the present invention relates to a vector system that is capable of delivering one or more NOIs to a target cell in vivo or in vitro. It is possible to manipulate the viral genome so that viral genes are replaced or supplemented with one or more NOIs which may be heterologous NOIs.

The term "heterologous" refers to a nucleic acid or protein sequence linked to a nucleic acid or protein sequence to which it is not naturally linked.

In the present invention, the term NOI includes any suitable nucleotide sequence, which need not necessarily be a complete naturally occurring DNA or RNA sequence. Thus, the NOI can be, for example, a synthetic RNA/DNA sequence, a recombinant RNA/DNA sequence (i.e. prepared by use of recombinant DNA techniques), a cDNA sequence or a partial genomic DNA sequence, including combinations thereof. The sequence need not be a coding region. If it is a coding region, it need not be an entire coding region. In addition, the RNA/DNA sequence can be in a sense orientation or in an anti-sense orientation. Preferably, it is in a sense orientation. Preferably, the sequence is, comprises, or is transcribed from cDNA.

The retroviral vector genome may generally comprise LTRs at the 5' and 3' ends, suitable insertion sites for inserting one or more NOI(s), and/or a packaging signal to enable the genome to be packaged into a vector particle in a producer cell. There may even be suitable primer binding sites and integration sites to allow reverse transcription of the vector RNA to DNA, and integration of the proviral DNA into the target cell genome. In a preferred embodiment, the retroviral vector particle has a reverse transcription system (compatible reverse transcription and primer binding sites) and an integration system (compatible integrase and integration sites).

The NOI may encode a protein of interest ("POI"). In this way, the vector delivery system could be used to examine the effect of expression of a foreign gene on the target cell. For example, the retroviral delivery system could be used to screen a cDNA library for a particular effect on the brain, motor neuron or CSF.

In accordance with the present invention, suitable NOIs include those that are of therapeutic and/or diagnostic application such as, but not limited to: sequences encoding cytokines, chemokines, hormones, antibodies, anti-oxidant molecules, engineered immunoglobulin-like molecules, single chain antibodies, fusion proteins, enzymes, immune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA,siRNA, shRNA, microRNA, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a tumor suppresser protein and growth factors, membrane proteins, vasoactive proteins and peptides, anti-viral proteins and ribozymes, and derivatives thereof (such as with an associated reporter group). The NOIs may also encode pro-drug activating enzymes.

The expression products encoded by the NOIs may be proteins which are secreted from the cell. Alternatively the NOI expression products are not secreted and are active within the cell. In either event, it is preferred for the NOI expression product to demonstrate a bystander effect or a distant bystander effect; that is the production of the expression product in one cell leading to the killing of additional, related cells, either neighboring or distant (e.g. metastatic), which possess a common phenotype.

The NOI or its expression product may act to modulate the biological activity of a compound or a pathway. As used herein the term "modulate" includes for example enhancing or inhibiting biological activity. Such modulation may be direct (e.g. including cleavage of, or competitive binding of another substance to a protein) or indirect (e.g. by blocking the initial production of a protein).

The NOI may be capable of blocking or inhibiting the expression of a gene in the target cell. For example, the NOI may be an antisense sequence. The inhibition of gene expression using antisense technology is well known.

The NOI or a sequence derived therefrom may be capable of "knocking out" the expression of a particular gene in the target cell. There are several "knock out" strategies known in the art. For example, the NOI may be capable of integrating in the genome of a neuron so as to disrupt expression of the particular gene. The NOI may disrupt expression by, for example, introducing a premature stop codon, by rendering the downstream coding sequence out of frame, or by affecting the capacity of the encoded protein to fold (thereby affecting its function).

Alternatively, the NOI may be capable of enhancing or inducing ectopic expression of a gene in the target cell. The NOI or a sequence derived therefrom may be capable of "knocking in" the expression of a particular gene.

In one preferred embodiment, the NOI encodes a ribozyme. Ribozymes are RNA molecules that can function to catalyze specific chemical reactions within cells without the obligatory participation of proteins. For example, group I ribozymes take the form of introns which can mediate their own excision from self-splicing precursor RNA. Other ribozymes are derived from self-cleaving RNA structures which are essential for the replication of viral RNA molecules. Like protein enzymes, ribozymes can fold into secondary and tertiary structures that provide specific binding sites for substrates as well as cofactors, such as metal ions. Examples of such structures include hammerhead, hairpin or stem-loop, pseudoknot and hepatitis delta antigenomic ribozymes.

Each individual ribozyme has a motif which recognizes and binds to a recognition site in a target RNA. This motif takes the form of one or more "binding arms" but generally two binding arms. The binding arms in hammerhead ribozymes are the flanking sequences Helix I and Helix III which flank Helix II. These can be of variable length, usually between 6 to 10 nucleotides each, but can be shorter or longer. The length of the flanking sequences can affect the rate of cleavage. For example, it has been found that reducing the total number of nucleotides in the flanking sequences from 20 to 12 can increase the turnover rate of the ribozyme cleaving a HIV sequence, by 10-fold (Goodchild 1991 Arch. Biochem. Biophys. 284:386-391). A catalytic motif in the ribozyme Helix II in hammerhead ribozymes cleaves the target RNA at a site which is referred to as the cleavage site. Whether or not a ribozyme will

cleave any given RNA is determined by the presence or absence of a recognition site for the ribozyme containing an appropriate cleavage site.

Each type of ribozyme recognizes its own cleavage site. The hammerhead ribozyme cleavage site has the nucleotide base triplet GUX directly upstream where G is guanine, U is uracil and X is any nucleotide base. Hairpin ribozymes have a cleavage site of BCUGNYR, where B is any nucleotide base other than adenine, N is any nucleotide, Y is cytosine or thymine and R is guanine or adenine. Cleavage by hairpin ribozymes takes places between the G and the N in the cleavage site.

More details on ribozymes may be found in "Molecular Biology and Biotechnology" (Ed. RA Meyers, 1995, VCH Publishers Inc. pp 831-8320 and in "Retroviruses" (Ed. JM Coffin et al., 1997, Cold Spring Harbor Laboratory Press pp 683).

Expression of the ribozyme may be induced in all cells, but will only exert an effect in those in which the target gene transcript is present.

Alternatively, instead of preventing the association of the components directly, the substance may suppress the biologically available amount of a polypeptide of the invention. This may be by inhibiting expression of the component, for example at the level of transcription, transcript stability, translation or post-translational stability. An example of such a substance would be antisense RNA or double-stranded interfering RNA sequences which suppresses the amount of mRNA biosynthesis.

In another preferred embodiment, the NOI comprises a siRNA. Post-transcriptional gene silencing (PTGS) mediated by double-stranded RNA (dsRNA) is a conserved cellular defense mechanism for controlling the expression of foreign genes. It is thought that the random integration of elements such as transposons or viruses causes the expression of dsRNA which activates sequence-specific degradation of homologous single-stranded mRNA or viral genomic RNA. The silencing effect is known as RNA interference (RNAi). The mechanism of RNAi involves the processing of long dsRNAs into duplexes of 21-25 nucleotide (nt) RNAs. These products are called small interfering or silencing RNAs (siRNAs) which are the sequence- specific mediators of mRNA degradation. In differentiated mammalian cells dsRNA >30bp has been found to activate the interferon response leading to shut-down of protein synthesis and nonspecific mRNA degradation. However this response can be bypassed by using around 21nt siRNA duplexes allowing gene function to be analyzed in cultured mammalian cells.

In one embodiment an RNA polymerase III promoter, e.g., U6, whose activity is regulated by the presence of tetracycline may be used to regulate expression of the siRNA.

In a further embodiment the NOI comprises double-stranded interfering RNA in the form of a hairpin. The short hairpin may be expressed from a single promoter, e.g., U6. In an alternative embodiment an effective RNAi may be mediated by incorporating two promoters, e.g., U6 promoters, one expressing a region of sense and the other the reverse complement of the same sequence of the target. In a further embodiment effective or double-stranded interfering RNA may be mediated by using two opposing promoters to transcribe the sense and antisense regions of the target from the forward and complementary strands of the expression cassette.

In another embodiment the NOI may encode a short RNA which may act to redirect splicing ('exon-skipping') or polyadenylation or to inhibit translation.

The NOI may also be an antibody. As used herein, "antibody" includes a whole immunoglobulin molecule or a part thereof or a bioisostere or a mimetic thereof or a derivative thereof or a combination thereof. Examples of a part thereof include: Fab, F(ab)' ₂ , and Fv. Examples of a bioisostere include single chain Fv (ScFv) fragments, chimeric antibodies, bifunctional antibodies.

Transduced target cells which express a particular gene, or which lack the expression of a particular gene have applications in drug discovery and target validation. The expression system could be used to determine which genes have a desirable effect on target cells, such as those genes or proteins which are able to prevent or reverse the triggering of apoptosis in the cells. Equally, if the inhibition or blocking of expression of a particular gene is found to have an undesirable effect on the target cells, this may open up possible therapeutic strategies which ensure that expression of the gene is not lost.

The present invention may therefore be used in conjunction with disease models, such as experimental allergic encephalomyelitis, which is the animal model of multiple sclerosis, and experimental autoimmune neuritis which is the animal model of acute and chronic inflammatory demyelinating polyneuropathy. Other disease models are known to those skilled in the art.

An NOI delivered by the vector delivery system may be capable of immortalizing the target cell. A number of immortalization techniques are known in the art (see for example Katakura et al. 1998 Methods Cell Biol. 57:69-91).

The term "immortalized" is used herein to cells capable of growing in culture for greater than 10 passages, which may be maintained in continuous culture for greater than about 2 months.

Immortalized motor and sensory neurons and brain cells are useful in experimental procedures, screening programs and in therapeutic applications. For example, immortalized dopaminergic neurons may be used for transplantation, for example to treat Parkinson's disease.

An NOI delivered by the vector delivery system may be a selection gene, or a marker gene. Many different selectable markers have been used successfully in retroviral vectors. These are reviewed in "Retroviruses" (1997, Cold Spring Harbor Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 444) and include, but are not limited to, the bacterial neomycin and hygromycin phosphotransferase genes which confer resistance to G418 and hygromycin respectively; a mutant mouse dihydrofolate reductase gene which confers resistance to methotrexate; the bacterial gpt gene which allows cells to grow in medium containing mycophenolic acid, xanthine and aminopterin; the bacterial hisD gene which allows cells to grow in medium without histidine but containing histidinol; the multidrug resistance gene (mdr) which confers resistance to a variety of drugs; and the bacterial genes which confer resistance to puromycin or phleomycin. All of these markers are dominant selectable and allow chemical selection of most cells expressing these genes.

An NOI delivered by the vector delivery system may be a therapeutic gene - in the sense that the gene itself may be capable of eliciting a therapeutic effect or it may encode a product that is capable of eliciting a therapeutic effect.

The term "mimetic" relates to any chemical which may be a peptide, polypeptide, antibody or other organic chemical which has the same binding specificity as the antibody.

The term "derivative" as used herein includes chemical modification of an antibody. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group.

MUTANTS, VARIANTS. HOMOLOGUES AND FRAGMENTS

The term "wild-type" is used to mean a polypeptide having a primary amino acid sequence which is identical with the native protein (i.e., the viral protein).

The term "mutant" is used to mean a polypeptide having a primary amino acid sequence which differs from the wild-type sequence by one or more amino acid additions, substitutions or

deletions. A mutant may arise naturally, or may be created artificially (for example by site- directed mutagenesis). Preferably the mutant has at least 90% sequence identity with the wild-type sequence. Preferably the mutant has 20 mutations or less over the whole wild-type sequence. More preferably the mutant has 10 mutations or less, most preferably 5 mutations or less over the whole wild-type sequence.

The term "variant" is used to mean a naturally occurring polypeptide which differs from a wild-type sequence. A variant may be found within the same viral strain (i.e. if there is more than one isoform of the protein) or may be found within a different strain. Preferably the variant has at least 90% sequence identity with the wild type sequence. Preferably the variant has 20 mutations or less over the whole wild-type sequence. More preferably the variant has 10 mutations or less, most preferably 5 mutations or less over the whole wild-type sequence.

Here, the term "homologue" means an entity having a certain homology with the wild-type amino acid sequence and the wild-type nucleotide sequence. Here, the term "homology" can be equated with "identity".

In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence. Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.

Percent homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with

the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalizing unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al. 1984 Nucleic Acids Res. 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. 1999 (ibid) - Chapter 18), FASTA (Atschul et al. 1990 J. MoI. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. 1999 (ibid), pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2

Sequences is also available for comparing protein and nucleotide sequence (see 1999 FEMS Microbiol Lett. 174(2):247-50; 1999 FEMS Microbiol. Lett. 177(1): 187-8).

Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to Table 2. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

Table 2

The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

Replacements may also be made by unnatural amino acids include; alpha* and alpha- disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-I- phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyric acid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-amino caproic acid , 7-amino heptanoic acid*, L-methionine sulfone* ^* , L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline ^# , L thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl- Phe*, L-Phe (4-amino) ^# , L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1,2,3,4- tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid ^# and L-Phe (4-benzyl)*. The notation * has been utilised for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al. 1992 PNAS 89(20):9367-9371 and Horwell 1995 Trends Biotechnol. 13(4): 132-134.

The term "fragment" indicates that the polypeptide comprises a fraction of the wild-type amino acid sequence. It may comprise one or more large contiguous sections of sequence or a plurality of small sections. The polypeptide may also comprise other elements of sequence, for example, it may be a fusion protein with another protein. Preferably the polypeptide comprises at least 50%, more preferably at least 65%, most preferably at least 80% of the wild-type sequence.

With respect to function, the mutant, variant, homologue or fragment should be capable of transducing at least part of the brain, a motor neuron or cerebrospinal fluid (CSF when used to pseudotype an appropriate vector.

The mutant, variant, homologue or fragment should alternatively or in addition, be capable of conferring the capacity for retrograde transport on the vector system.

The vector delivery system used in the present invention may comprise nucleotide sequences that can hybridize to the nucleotide sequence presented herein (including complementary sequences of those presented herein). In a preferred aspect, the present invention covers nucleotide sequences that can hybridize to the nucleotide sequence of the present invention under stringent conditions (e.g. 65 ⁰ C and 0.1 X SSC) to the nucleotide sequence presented herein (including complementary sequences of those presented herein).

A potential advantage of using the rabies glycoprotein is the detailed knowledge of its toxicity to humans and other animals due to the extensive use of rabies vaccines. In particular, phase 1 clinical trials have been reported on the use of rabies glycoprotein expressed from canarypox recombinant virus as a human vaccine (Fries et al. 1996 Vaccine 14:428-434); these studies concluded that the vaccine was safe for use in humans.

mjRNA

MicroRNAs are a large group of small RNAs produced naturally in organisms, at least some of which regulate the expression of target genes. Founding members of the miRNA family are let-7 and lin-4. The let-7 gene encodes a small, highly conserved RNA species that regulates the expression of endogenous protein-coding genes during worm development. The active RNA species is transcribed initially as an approximately 70 nucleotide precursor, which is post- transcriptionally processed into a mature double stranded approximately 21 nucleotide form. Both let-7 and lin-4 are transcribed as hairpin RNA precursors which are processed to their mature forms by the Dicer enzyme.

The retroviral vector of the invention preferably comprises a miRNA target site. A "miRNA target site" is a sequence to which an endogenous miRNA binds. The miRNA target site is complementary to the endogenous miRNA. Expression of one or more NOIs can be reduced in cells containing miRNAs by including a sequence encoding a miRNA target site in a vector comprising the NOI. Binding of the miRNA to the miRNA target site suppresses expression of the one or more NOIs. If the miRNA is tissue-specific, NOI expression is suppressed only in cell types comprising endogenous miRNAs that bind to the miRNA target site, while the NOI is expressed in cell types that do not express such miRNAs (i.e. target cells).

Three miRNAs, miR-lb/d, miR-133, and miR-206, having tissue-biased expression profiles in muscle have been identified. In a preferred embodiment, the present invention utilizes these miRNAs in a lentiviral-mediated transgene delivery system to increase specificity of transgene expression in motor neuron populations, while inhibiting expression in transduced muscle cells following intramuscular delivery.

RETROVIRUS PRODUCTION

The vector system of the present invention is preferably a viral vector system, more preferably a retroviral vector system, and most preferably a lentiviral vector system.

Retroviral vector systems have been proposed as a delivery system for inter alia the transfer of a nucleotide sequence of interest (NOI) to one or more sites of interest. The transfer can occur in vitro, ex vivo, in vivo, or combinations thereof. Retroviral vector systems have even been exploited to study various aspects of the retrovirus life cycle, including receptor usage, reverse transcription and RNA packaging (reviewed by Miller 1992 Curr. Top. Microbiol. Immunol. 158:1-24).

As used herein the term "vector system" may also include a vector particle capable of transducing a recipient cell with an NOI.

A vector particle includes the following components: a vector genome, which may contain one or more NOIs, a nucleocapsid encapsidating the nucleic acid, and a membrane surrounding the nucleocapsid.

The term "nucleocapsid" refers to at least the group specific viral core proteins (gag) and the viral polymerase (pol) of a retrovirus genome. These proteins encapsidate the packagable sequences and are themselves further surrounded by a membrane containing an envelope glycoprotein.

Once within the cell, the RNA genome from a retroviral vector particle is reverse transcribed into DNA and integrated into the DNA of the recipient cell.

The term "vector genome" refers both to the RNA construct present in the retroviral vector particle and the integrated DNA construct. The term also embraces a separate or isolated DNA construct capable of encoding such an RNA genome. A retroviral or lentiviral genome should comprise at least one component part derivable from a retrovirus or a lentivirus. The term "derivable" is used in its normal sense as meaning a nucleotide sequence or a part thereof which need not necessarily be obtained from a virus such as a lentivirus but instead could be derived therefrom. By way of example, the sequence may be prepared synthetically or by use of recombinant DNA techniques. Preferably the genome comprises apsi region (or an analogous component which is capable of causing encapsidation).

The viral vector genome is preferably "replication defective" by which we mean that the genome does not comprise sufficient genetic information alone to enable independent replication to produce infectious viral particles within the recipient cell. In a preferred embodiment, the genome lacks a functional env, gag or pol gene. If a highly preferred embodiment the genome lacks env, gag and pol genes.

The viral vector genome may comprise some or all of the LTRs. Preferably the genome comprises at least part of the LTRs or an analogous sequence which is capable of mediating proviral integration, and transcription. The sequence may also comprise or act as an enhancer- promoter sequence.

It is known that the separate expression of the components required to produce a retroviral vector particle on separate DNA sequences co-introduced into the same cell will yield retroviral

particles carrying defective retroviral genomes that carry therapeutic genes {e.g. reviewed by Miller 1992 (supra)). This cell is referred to as the producer cell.

There are two common procedures for generating producer cells. In one, the sequences encoding retroviral gag, pol and env proteins are introduced into the cell and stably integrated into the cell genome; a stable cell line is produced which is referred to as the packaging cell line. The packaging cell line produces the proteins required for packaging retroviral RNA but it cannot bring about encapsidation due to the lack of z.psi region. However, when a vector genome (having a psi region) is introduced into the packaging cell line, the helper proteins can package the /rø-positive recombinant vector RNA to produce the recombinant virus stock. This can be used to transduce the NOI into recipient cells. The recombinant virus whose genome lacks all genes required to make viral proteins can infect only once and cannot propagate. Hence, the NOI is introduced into the host cell genome without the generation of potentially harmful retrovirus. A summary of the available packaging lines is presented in "Retroviruses" (1997, Cold Spring Harbor Laboratory Press, Eds: JM Coffin, SM Hughes, HE Varmus pp 449).

The second approach is to introduce the three different DNA sequences that are required to produce a retroviral vector particle i.e. the env coding sequences, the gag-pol coding sequence and the defective retroviral genome containing one or more NOIs into the cell at the same time by transient transfection and the procedure is referred to as transient triple transfection (Landau and Littman 1992; Pear et al. 1993). The triple transfection procedure has been optimized (Soneoka et al. 1995 Nucleic Acids Res. 23(4):628-33; Finer et al. 1994). WO 94/29438 describes the production of producer cells in vitro using this multiple DNA transient transfection method. WO 97/27310 describes a set of DNA sequences for creating retroviral producer cells either in vivo or in vitro for re-implantation.

The components of the viral system which are required to complement the vector genome may be present on one or more "producer plasmids" for transfecting into cells.

In a preferred embodiment, the viral vector genome is incapable of encoding the proteins gag, pol and env. Preferably the viral vector system comprises one or more producer plasmids encoding env, gag and pol, for example, one producer plasmid encoding env and one encoding gag-pol. Preferably the gag-pol sequence is codon optimized for use in the particular producer cell.

The present invention also provides a producer cell expressing the vector genome and the producer plasmid(s) capable of producing a retroviral vector system of the present invention.

Preferably the retroviral vector system of the present invention is a self-inactivating (SIN) vector system.

By way of example, self-inactivating retroviral vector systems have been constructed by deleting the transcriptional enhancers or the enhancers and promoter in the U3 region of the 3' LTR. After a round of vector reverse transcription and integration, these changes are copied into both the 5' and the 3' LTRs producing a transcriptionally inactive provirus. However, any promoter(s) internal to the LTRs in such vectors will still be transcriptionally active. This strategy has been employed to eliminate effects of the enhancers and promoters in the viral LTRs on transcription from internally placed genes. Such effects include increased transcription or suppression of transcription. This strategy can also be used to eliminate downstream transcription from the 3' LTR into genomic DNA. This is of particular concern in human gene therapy where it may be important to prevent the adventitious activation of an endogenous oncogene.

Preferably a recombinase assisted mechanism is used which facilitates the production of high titer regulated lentiviral vectors from the producer cells of the present invention.

As used herein, the term "recombinase assisted system" includes but is not limited to a system using the Cre recombinase / loxP recognition sites of bacteriophage Pl or the site-specific FLP recombinase of S. cerevisiae which catalyses recombination events between 34 bp FLP recognition targets (FRTs).

The site-specific FLP recombinase of 5. cerevisiae which catalyses recombination events between 34 bp FLP recognition targets (FRTs) has been configured into DNA constructs in order to generate high level producer cell lines using recombinase-assisted recombination events (Karreman et al. 1996 Nucleic Acids Res. 24:1616-1624). A similar system has been developed using the Cre recombinase / loxP recognition sites of bacteriophage Pl (see PCT/GBOO/03837; Vanin et al. 1997 J. Virol. 71:7820-7826). This was configured into a lentiviral genome such that high titer lentiviral producer cell lines were generated.

By using producer/packaging cell lines, it is possible to propagate and isolate quantities of retroviral vector particles {e.g. to prepare suitable titers of the retroviral vector particles) for

subsequent transduction of, for example, a site of interest (such as adult brain tissue). Producer cell lines are usually better for large scale production or vector particles.

Transient transfection has numerous advantages over the packaging cell method. In this regard, transient transfection avoids the longer time required to generate stable vector-producing cell lines and is used if the vector genome or retroviral packaging components are toxic to cells. If the vector genome encodes toxic genes or genes that interfere with the replication of the host cell, such as inhibitors of the cell cycle or genes that induce apoptosis, it may be difficult to generate stable vector-producing cell lines, but transient transfection can be used to produce the vector before the cells die. Also, cell lines have been developed using transient infection that produce vector titer levels that are comparable to the levels obtained from stable vector- producing cell lines (Pear et al. 1993 PNAS 90:8392-8396).

Producer cells/packaging cells can be of any suitable cell type. Producer cells are generally mammalian cells but can be, for example, insect cells.

As used herein, the term "producer cell" or "vector producing cell" refers to a cell which contains all the elements necessary for production of retroviral vector particles.

Preferably, the producer cell is obtainable from a stable producer cell line.

Preferably, the producer cell is obtainable from a derived stable producer cell line.

Preferably, the producer cell is obtainable from a derived producer cell line.

As used herein, the term "derived producer cell line" is a transduced producer cell line which has been screened and selected for high expression of a marker gene. Such cell lines support high level expression from the retroviral genome. The term "derived producer cell line" is used interchangeably with the term "derived stable producer cell line" and the term "stable producer cell line".

Preferably the derived producer cell line includes but is not limited to a retroviral and/or a lenti viral producer cell.

Preferably the derived producer cell line is an HIV or EIAV producer cell line, more preferably an EIAV producer cell line.

In order to minimize potential for expression of the transgene in producer cells, such as 293T cells, the cloning of transgenes into the vectors has been designed in such a way that the first NOI is out of frame with respect to any upstream ORFs.

Preferably the envelope protein sequences, and nucleocapsid sequences are all stably integrated in the producer and/or packaging cell. However, one or more of these sequences could also exist in episomal form and gene expression could occur from the episome.

As used herein, the term "packaging cell" refers to a cell which contains those elements necessary for production of infectious recombinant virus which are lacking in the RNA genome. Typically, such packaging cells contain one or more producer plasmids which are capable of expressing viral structural proteins (such as codon optimized gag-pol and env) but they do not contain a packaging signal.

The term "packaging signal" which is referred to interchangeably as "packaging sequence" or "psi" is used in reference to the non-coding, czs-acting sequence required for encapsidation of retroviral RNA strands during viral particle formation. In HIV-I, this sequence has been mapped to loci extending from upstream of the major splice donor site (SD) to at least the gag start codon.

Packaging cell lines suitable for use with the above-described vector constructs may be readily prepared (see also WO 92/05266), and utilized to create producer cell lines for the production of retroviral vector particles. As already mentioned, a summary of the available packaging lines is presented in the reference entitled "Retroviruses" (supra).

Also as discussed above, simple packaging cell lines, comprising a provirus in which the packaging signal has been deleted, have been found to lead to the rapid production of undesirable replication competent viruses through recombination. In order to improve safety, second generation cell lines have been produced wherein the 3'LTR of the provirus is deleted. In such cells, two recombinations would be necessary to produce a wild type virus. A further improvement involves the introduction of the gag-pol genes and the env gene on separate constructs so-called third generation packaging cell lines. These constructs are introduced sequentially to prevent recombination during transfection.

Preferably, the packaging cell lines are second generation packaging cell lines.

Preferably, the packaging cell lines are third generation packaging cell lines.

In these split-construct, third generation cell lines, a further reduction in recombination may be achieved by changing the codons. This technique, based on the redundancy of the genetic code, aims to reduce homology between the separate constructs, for example between the regions of overlap in the gag-pol and env open reading frames.

The packaging cell lines are useful for providing the gene products necessary to encapsidate and provide a membrane protein for a high titer vector particle production. The packaging cell may be a cell cultured in vitro such as a tissue culture cell line. Suitable cell lines include but are not limited to mammalian cells such as murine fibroblast derived cell lines or human cell lines. Preferably the packaging cell line is a human cell line, such as for example: HEK293, 293-T, TE671, HT1080.

Alternatively, the packaging cell may be a cell derived from the individual to be treated such as a monocyte, macrophage, blood cell or fibroblast. The cell may be isolated from an individual and the packaging and vector components administered ex vivo followed by re-administration of the autologous packaging cells.

In more detail, the packaging cell may be an in vivo packaging cell in the body of an individual to be treated or it may be a cell cultured in vitro such as a tissue culture cell line. Suitable cell lines include mammalian cells, such as murine fibroblast derived cell lines or human cell lines. Preferably the packaging cell line is a human cell line, such as 293 cell line, HEK293, 293-T, TE671, or HTl 080.

Alternatively, the packaging cell may be a cell derived from the individual to be treated such as a monocyte, macrophage, stem cells, blood cell or fibroblast, etc. The cell may be isolated from an individual and the packaging and vector components administered ex vivo followed by re-administration of the autologous packaging cells. Alternatively the packaging and vector components may be administered to the packaging cell in vivo. Methods for introducing lentiviral packaging and vector components into cells of an individual are known in the art. For example, one approach is to introduce the different DNA sequences that are required to produce a lentiviral vector particle e.g. the env coding sequence, the gag-pol coding sequence and the defective lentiviral genome into the cell simultaneously by transient triple transfection (Landau and Littman 1992 /. Virol. 66:5110; Soneoka et al. 1995 Nucleic Acids Res. 23:628- 633).

It is highly desirable to use high-titer virus preparations in both experimental and practical applications. Techniques for increasing viral titer include using a psi plus packaging signal as discussed above and concentration of viral stocks.

As used herein, the term "high titer" means an effective amount of a retroviral vector or particle which is capable of transducing a target site such as a cell.

As used herein, the term "effective amount" means an amount of a regulated retroviral or lentiviral vector or vector particle which is sufficient to induce expression of the NOIs at a target site.

A high-titer viral preparation for a producer/packaging cell is usually of the order of 10 ⁵ to 10 ⁷ t.u. per ml. (The titer is expressed in transducing units per ml (LuVmI) as titered on a standard D17 cell line). For transduction in tissues such as the DRG, it is necessary to use very small volumes, so the viral preparation is concentrated by ultracentrifugation. The resulting preparation should have at least 10 ⁸ t.uVml, preferably from 10 ^s to 10 ⁹ t.uVml, more preferably at least 10 ⁹ t.u./ml.

In one embodiment the vector configurations of the present invention use as their production system, three transcription units expressing a genome, the gag-pol components and an envelope. The envelope expression cassette may include one of a number of envelopes such as VSV-G or various murine retrovirus envelopes such as 4070A.

Conventionally these three cassettes would be expressed from three plasmids transiently transfected into an appropriate cell line such as 293T or from integrated copies in a stable producer cell line. An alternative approach is to use another virus as an expression system for the three cassettes, for example baculovirus or adenovirus. These are both nuclear expression systems. To date the use of a poxvirus to express all of the components of a lentiviral vector system has not been described. In particular, given the unusual codon usage of lentiviruses and their requirement for RNA handling systems such as the rev/RRE system

DISEASES

The vector system used in the present invention is particularly useful in treating and/or preventing a disease which is associated with the death or impaired function of cells of the nervous system, such as neurons, or glial cells. Thus, the vector system is useful in treating and/or preventing neurodegenerative diseases.

In particular, the vector system used in the present invention may be used to treat and/or prevent a disease which is associated with the death or impaired function of motor or sensory neurons.

Diseases which may be treated include, but are not limited to: pain; movement disorders such as Parkinson's disease, motor neuron diseases including amyotrophic lateral sclerosis (ALS or Lou Gehrig's Disease) and Huntington's disease; Alzheimer's Disease; Spinal Muscle

Atrophy (SMA) and Lysosomal Storage Diseases. In particularly preferred embodiments, the vector system of the invention is used to treat ALS or SMA

Amyotrophic lateral sclerosis (ALS) is a degenerative disorder of motor neurons with a yearly incidence of 1-2 per 100,000. It is characterized by degeneration of motor neurons in the spinal cord, brain stem and motor cortex which leads to wasting and weakness of limb, bulbar and respiratory muscles. Approximately 5-10% of ALS is familial. Genes whose mutations or haplotypes are thought to play a role in disease predisposition include SODl, ALS2 and VEGF (Lambrechts et al. 2003 Nature Genetics 34(4):383-94; Oosthuyse et al. 2001 Nature Genetics 28:131-138).

In particular, the vector system used in the present invention is useful in treating and/or preventing ALS. In this embodiment, the NOI may be capable of knockdown of SODl . Other NOI(s) may encode molecules which prevent apoptosis and therefore prevent cells from dying. Suitable molecules include XIAP and NAIP. Alternatively, NOI(s) may encode targets for pain modulation, neurotrophic molecules to provide neuroprotection or molecules to stimulate regeneration such as IGF-I, GDNF, NGF, BDNF, VEGF, RARβ2, IL-2 and cardiotrophin (CTl).

Spinal Muscular Atrophy (SMA) is a disease of the anterior horn cells and is an autosomal recessive disease. Anterior horn cells are located in the spinal cord. SMA affects the voluntary muscles for activities such as crawling, walking, head and neck control and swallowing. Categories of SMA include: Type I SMA also called Werdnig-Hoffmann Disease, Type II, Type III, often referred to as Kugelberg-Welander or Juvenile Spinal Muscular Atrophy, Type IV (Adult Onset) and Adult Onset X-Linked SMA also known as Kennedy's Syndrome or Bulbo-Spinal Muscular Atrophy. SMA is a motor neuron disease in humans and its most severe form causes death by the age of 2 years. It is caused by mutations in the telomeric survival motor neuron gene, SMNl. In particular, the vector system used in the present invention is useful in treating and/or preventing SMA. In this embodiment, the NOI may be capable of encoding a gene for replacement of defective SMNl gene. Other NOI(s) may encode molecules which prevent apoptosis and therefore prevent cells from dying. Suitable molecules include XIAP and NAIP. Alternatively, NOI(s) may encode neurotrophic molecules which stimulate regeneration such as IGF-I, GDNF, neurotrophin-3 (NT-3), VEGF and cardiotrophin (CTl).

In particular, in a preferred embodiment for treating motor neuron diseases, the vector system is a lentiviral vector system because advantageously with the use of a lentiviral vector

system having a rabies G pseudotype, one achieves high efficiency retrograde transport and long term expression. While both adenovirus and HSV and even AAV (to a lesser extent) do get retrogradely transported, the lentiviral vector system having a rabies G pseudotype achieves high efficiency retrograde transport through the selective transduction of neurons. Advantageously, lentiviral vectors pseudotyped with rabies G specifically target motor neurons with high efficiency. Moreover, the use of lentiviral vectors avoids the toxicity issues common to the use of adenovirus and HSV, for example.

It is a further advantage of a lentiviral vector system pseudotyped with a rabies glycoprotein G that retrograde transport occurs through the intramuscular route with little to no transduction of adult muscle cells (Mazarakis et al. 2001 Human Molec. Genet. 10:2109-2121) thereby exhibiting the selectivity necessary for efficient transduction of motor neurons, whereas the use of AAV may not be so selective in that transduction of motor neurons also results in long-lasting expression in the muscle (Lu et al. 2003 Neurosci. Res. 45(l):33~40). Moreover, the incorporation of muscle- specific miRNAs into the vector system of the invention further inhibits transgene expression in muscle cells.

PHARMACEUTICAL COMPOSITIONS

The present invention also provides a pharmaceutical composition for treating an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of the retroviral vector of the present invention comprising one or more deliverable therapeutic and/or diagnostic NOI(s) or a viral particle produced by or obtained from same.

The methods and pharmaceutical compositions of the invention may be used to treat a human or animal subject. Preferably the subject is a mammalian subject. More preferably the subject is a human. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.

The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s),

and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system).

Where appropriate, the pharmaceutical compositions can be administered by any one or more of: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents, or they can be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

The vector system used in the present invention may conveniently be administered by direct injection into the patient. Preferably, the system is injected into a given area, and the target area transduced by retrograde transport of the vector system. Intramuscular injection is particularly preferred as the least invasive method of treatment.

TRANSPORT

The present invention provides the use of a vector system to transduce a target site, wherein the vector system travels to the target site by retrograde transport.

A virus particle may travel in the same direction as a nerve impulse, i.e. from the cell body, along the axon to the axon terminals. This is known as anterograde transport.

The present inventors have shown that certain vector systems are transported in a retrograde manner, in the opposite direction of anterograde transport. Retrograde transport (or transfer) of a vector means that it is taken up by the axon terminals and travels toward the cell body. Whilst the precise mechanism of retrograde transport is unknown it is thought to involve transport of the whole viral particle, possibly in association with an internalized receptor.

The movement of membranous organelles at 50-200 mm per day toward the synapse (anterograde) or back to the cell body (retrograde) occurs via "fast transport" (Hirokawa 1997 Curr. Opin. Neurobiol. 7(5):605-614). The fact that the present vector systems can be

specifically transported in this manner (as demonstrated herein) suggests that the env protein may be involved.

HSV, adenovirus and hybrid HSV/adeno-associated virus vectors have all been shown to be transported in a retrograde manner in the brain (Horellou and Mallet 1997 MoI. Neurobiol. 15(2):241-256; Ridoux et al. 1994 Brain Res. 648:171-175; Constantini et al. 1999 Human Gene Ther. 10:2481-2494). Injection of an adenoviral vector system expressing glial cell line derived neurotrophic factor (GDNF) into rat striatum allows expression in both dopaminergic axon terminals and cell bodies via retrograde transport (Horellou and Mallet 1997 (supra); Bilang- Bleuel et al. 1997 PNAS USA 94:8818-8823).

Retrograde transport can be detected by a number of mechanisms known in the art. In the present examples, a vector system expressing a heterologous gene is injected into the striatum, and expression of the gene is detected in the substantia nigra. It is clear that retrograde transport along the neurons which extend from the substantia nigra to the basal ganglia is responsible for this phenomenon. It is also known to monitor labelled proteins or viruses and directly monitor their retrograde movement using real time confocal microscopy (Hirokawa 1997 (supra)).

By retrograde transport, it is possible to get expression in both the axon terminals and the cell bodies of transduced neurons. These two parts of the cell may be located in distinct areas of the nervous system. Thus, a single administration (for example, injection) of the vector system of the present invention may transduce many distal sites.

The present invention also provides the use of a vector system of the present invention to transduce a target site, which comprises the step of administration of the vector system to an administration site which is distant from the target site to achieve good penetration and biodistribution throughout the CNS. For example, administration to the one area of the brain may give rise to distribution of the NOI/POI in different parts of the brain and/different cell types.

The target site may be any site of interest. It may or may not be anatomically connected to the administration site. The target site may be capable of receiving vector from the administration site by axonal transport, for example anterograde or (more preferably) retrograde transport. For a given administration site, a number of potential target sites may exist which can be identified using tracers by methods known in the art (Ridoux et al. 1994 (supra)).

A target site is considered to be "distant from the administration" if it is (or is mainly) located in a different region from the administration site. The two sites may be distinguished by their spatial location, morphology and/or function. In a preferred embodiment of the present invention, the administration site is muscle, while the target site is the spinal cord.

Within a given target site, the vector system may transduce a target cell. The target cell may be a cell found in nervous tissue, such as a sensory or motor neuron, astrocyte, oligodendrocyte, microglia or ependymal cell. A "target cell" is a cell in which the NOI is expressed.

The target cell can be a cell found in the dorsal root ganglion (DRG), such as a glial cell. Alternatively, the target cell may be a cell found in the peripheral nervous tissue, such as a neuron, a glial cell or a Schwann cell.

In a particularly preferred embodiment, the NOI is delivered to the spinal cord by retrograde transport from muscle cells. In this case, the target cell may be, for example, a motor neuron, an interneuron, a glial cell, an astrocyte or an oligodendrocyte.

The target cell can also be a sensory neuron. Especially preferred is a sensory neuron either within a C fiber or an Aδ fiber. C fiber neurons are especially preferred.

Sensory neurons are pseudo-unipolar neurons having a single process which projects from the cell body. This process bifurcates to form terminals in the periphery (the peripheral branch) and in the grey matter of the spinal cord where they synapse with other neurons (the central branch).

If the NOI is delivered to the cell body of a sensory neuron in the DRG it can then travel (with or without modification) to the spinal cord via the central branch. For example, the vector system may deliver an NOI to the cell body of a sensory neuron. The NOI may be translated into a POI within the cell body and the POI delivered to the spinal cord via the central branch.

In this embodiment, the NOI or POI may, for example, be capable of modulating the activity or expression of a neurotransmitter, a neurotrophin (such as GDNF, BDNF, NT3, CTNF and nerve growth factor), an antiapoptotic factor, an ion channel and or a receptor such as RARβ2.

This method may be used for non-invasive access to the CNS, and so it is suitable for the treatment and/or prevention of any condition which affects the brain and/or spinal cord. These include conditions associated with motor neurons, such as motor neuron disease. For example,

ALS may be treatable with the use of anti-apoptotic factors. SMA (in neonates) may be preventable or treatable by replacing survival motor neuron gene 1, in order to avoid apoptosis. These also include other conditions associated with sensory neurons. For example encephalins may be used to regrow sensory neurons in conditions such as paraplegia.

The vector system is preferably administered by direct injection. Methods for injection into the brain (in particular the striatum) are well known in the art (Bilang-Bleuel et al. 1997 PNAS USA 94:8818-8823; Choi-Lundberg et al. 1998 Exp. _VeκrøZ.154:261-275; Choi-Lundberg et al. 1997 Science 275:838-841; and Mandel et al. 1997 PNAS USA 94:14083-14088). Stereotaxic injections may be given. In a particularly preferred embodiment, the vector system is delivered via intramuscular injection.

Various preferred features and embodiments of the present invention will now be described by way of non-limiting example.

EXAMPLES

Example 1. Construction of pseudotyped EIAV vectors

Vector Nomenclature pONYKZ is identical to pONY8.9NCZ except that the ampicillin reistance gene has been replaced by the kanamycin resistance gene. pONY8.9 series vectors have WPRE but no cPPT. These have been previously described in WO 03/064665 and WO 05/052171.

In the vector nomenclature:

"N" indicates the presence of neo,

"C" indicates the presence of CMV,

"G" indicates the presence of GFP,

"Z" indicates the presence of LacZ,

Construction ofpONYKZ pONYKZ is constructed from pONY8.9NCZ by substitution of the ampicillin resistance gene with the kanamycin resistance gene.

Construction of pONY8.9NCZ was previously described in WO 03/064665 and WO 05/052171.

Figure 3 shows a schematic diagram of pONYKZmiRX, where X is any miRNA target sequence.

Example 2. Cloning miRNA into pseudotyped EIAV vectors

Four tandem copies of a 22 or 23 -bp sequence with perfect complementarity to the muscle specific miR-ld, miR-133, or control miR-33 are cloned into the 3'-UTR of the EIAV backbone containing a LacZ expression cassette (pONYKZ). The control miR-33 is not enriched in muscle or neuronal tissue, and expression of the transgene in these tissue types with this construct should be constitutive.

Rabies-G pseudotyped EIAV vectors are constructed from each of these constructs and concentrated preps produced (pONYKZ-mild, pONYKZ-mil33, pONYKZ-mi33).

Example 3: Evaluating transgene expression

Transgene expression from vector constructs is evaluated by transducing a panel of relevant cell lines. Expression of the LacZ reporter gene is analyzed in these cell types by X-gal staining.

In vivo analysis is performed in wild-type mice. Vectors are injected intramuscularly into several muscle groups (hind limb, facial, tongue). Animals are sacrificed two weeks post injection and analysis of β-gal expression is performed on injected muscle groups, spinal cord and brain samples. Q-RT-PCR analysis of vector mRNA is to be performed in these tissue types.