WO2001089580A1

WO2001089580A1 - Systemic and cardiovascular transduction with lentiviral vectors

Info

Publication number: WO2001089580A1
Application number: PCT/EP2001/005876
Authority: WO
Inventors: Thierry Vandendriessche; Marinee Chuah; Luigi Naldini
Original assignee: Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw; D. Collen Research Foundation Vzw
Priority date: 2000-05-22
Filing date: 2001-05-22
Publication date: 2001-11-29
Also published as: AU2001278430A1

Abstract

The present invention relates to a method for obtaining systemic delivery and/or stable systemic transduction and/or stable cardiovascular transduction of a lentiviral vector, comprising introduction of said lentiviral vector into an animal.

Description

Systemic and cardiovascular transduction with lentiviral vectors

Gene therapy is a promising novel approach to treat a variety of disorders, including diseases such as cardiovascular, genetic, neurologic and infectious diseases and cancer. Various methods, both viral and non-viral, can be used to deliver the genes of interest into the cells to be targeted. Non-viral methods of gene delivery include methods such as cationic and cholesterol containing liposomes, peptide-lipid vectors, branched DNA-binding carbohydrates, bacteria and artificial chromosomes. The viral methods are using viral vectors based on viruses such as retroviruses, adenoviruses, adeno-associated viruses, herpes simplex virus and flaviviruses.

The retroviruses comprise a large group of viruses that infect primarily vertebrates. Lentiviruses are a subclass of retroviruses; they are however considerably more complicated than simple retroviruses. The subclass includes a variety of primate viruses such as Human Immunodeficiency Virus (HIV) and Simian Immunodeficiency Virus (SIV) and non-primate viruses such as Maedi Visna Virus (MW), Feline Immunodeficiency Virus (FIV), Equine Infectious Anemia Virus (EIAV), Caprine Arthrithis Encephalitis Virus (CAEV) and Bovine Immunodeficiency Virus (BIV). Unlike retroviruses, lentiviruses are able to infect both proliferating and non- proliferating cells (Lewis and Emerman, 1993). Therefore, lentiviral vectors, and particularly Human Immunodeficiency Virus (HIV) based lentiviral vectors are a promising tool for in vivo gene therapy. Indeed, unlike Moloney murine leukemia based (MoMLV) retroviral vectors, lentiviral vectors are in principle able to transduce dividing as well as non-dividing cells. Lentiviral vectors have demonstrated efficient and long-lasting gene transfer into a variety of human cells including cells such as nerve, liver, skeletal muscle (Blomer et al., 1997; Kafri et al., 1997; Miyoshi et al., 1997), bone marrow (White et a/.1999) and human corneal cells (Wang etal., 2000).

Gene therapy can be ex vivo or in vivo. In the ex vivo approach, biopsy cells or tissues are isolated, grown in culture, transduced with the gene therapy vector, and reintroduced in the body. In the in vivo approach, the vector is directly applied to the airways, or injected intratumor, intraperitoneal, subcutaneously, intramuscularly, intravenously, intra-arterialy or intraportally. For the in vivo approach with lentiviral vectors, the vector is normally injected in the target tissue where the expression is wanted, and a restricted expression pattern is obtained. Han et al. (1999) infused a lentiviral vector into the cochlea and obtained an expression pattern that was limited to the periphery of the perilymphatic space. After injection in the muscle, expression in the muscle was detected. In a similar way, Miyoshi et al. (1997) obtained expression in the retina after injection of the lentiviral vector in the subretinal space. Kafri et al. (1997) demonstrated that after injection of a lentiviral vector in the liver parenchyma, expression in the liver could be detected. In none of these cases, there was evidence for a further distribution of the lentiviral vector into other tissues. More recent studies indicate that non-activated, non-proliferating liver cells are refractory to lentiviral transduction (Park et al., 2000). In addition, non-activated T- or B-cells cannot be transduced with lentiviral vectors. Hence, some cells are relatively refractory to lentiviral transduction. In addition, the prior art shows no evidence for a further distribution of the lentiviral vector into the vasculature and transduction of cardiovascular target cells in vivo or ex vivo (e.g. vascular grafts). Hence, it cannot be predicted based on these observations that the cardiovascular system can be stably transduced with lentiviral vectors. Furthermore, direct in vivo delivery of Moloney-based retroviral vectors by intravenous injection in neonatal mice did not yield detectable gene transfer into the cardiovascular system (VandenDriessche et al., 1999).

Surprisingly, we found that intravenous injection of a lentiviral vector into neonatal mice lead to a stable and systemic delivery and transduction of the vector. Even more surprisingly, it could be demonstrated that the transduction was in the cardiovascular system.

It is one aspect of the invention to provide an easy and simple method to obtain systemic in vivo delivery of a lentiviral vector, whereby the method is comprising intravascular, preferably intravenous injection of said lentiviral vector.

It is another aspect of the invention to provide an easy and simple method to obtain stable and systemic in vivo transduction of animal cells, whereby the method is comprising intravascular, preferably intravenous injection of a lentiviral vector. Preferably, said lentiviral vector is injected into a neonatal animal. In one embodiment, the lentiviral vector is a VSV-G pseudotyped HIV derived lentiviral vector. It is important to note that systemic delivery does not necessarily imply systemic targeting, neither systemic expression of the gene of interest in the lentiviral vector. Even in the case of systemic delivery, it is known to a person skilled in the art that the targeting of the vector can be restricted by using specific targeting of said vector to the tissue of interest by modification of the envelope of the lentiviral vector. Moreover, some target tissues can be more susceptible to transduction than others, which may restrict the targeting pattern in a natural way. In addition, systemic targeting does not necessarily implies systemic expression. It is known to the person skilled in the art that the expression can be restricted with the help of a tissue specific promoter.

It is another aspect of the invention to provide a method to obtain stable systemic in vivo transduction of cardiovascular tissue of an animal, by intravascular, preferably intravenous injection of a lentiviral vector. Preferably, said vector is injected in neonatal animals. More preferably, said vector is a VSV-G pseudotyped HIV derived vector. Cardiovascular tissue, as defined here, comprises endothelial cells and/or smooth muscle cells.

It is still another aspect of the invention to provide a method to obtain transduction of the cardiovascular system, whereby the method is comprising introdution of a lentiviral vector. Preferably, the introduction is intravascular, preferably intravenous injection. Even more preferably, said lentiviral vector is a VSV-G pseudotyped HIV derived vector. One preferred embodiment is the ex vivo transduction of said lentiviral vector into vascular tissue obtained from said animal. Still another aspect of the invention is the use of said methods to treat diseases, such as cardiovascular diseases, genetic diseases, cancer, neurological disorders (Parkinson, Alzheimer), allergy (including but not limited to genetic tolerance induction) and infectious diseases (including but not limited to genetic vaccination).

Definitions

The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.

Stable transduction means that the transduction still can be detected at least 2 months after the introduction of the lentiviral vector. Detection of the vector can be directly, e.g. by hybridisation, or indirectly, by detection of the expression and/or activity of one of the transduced genes.

Systemic delivery means that the packaged lentiviral vector after injection at one place in the body is not only delivered to the tissue, adjacent to the place of injection, but is reaching other tissues and/or organs, at least liver, spleen, lung and heart, preferentially at least liver, spleen, lung, heart, brain and testis. Systemic delivery does not necessarily imply systemic transduction, which is dependent upon the ability of the vector particle to transduce the tissue where it is delivered.

Systemic transduction means that the transduction is not limited to specific organs, but at least can be detected in liver, spleen, lung and heart, preferentially in at least liver, spleen, lung, heart, brain and testis. Transduction as used here means the uptake of the lentiviral vector in the cell under a form that allows transcription and translation of a gene of interest, when said gene is placed under the control of the appropriate control signals. The control signals may be sequences such as a tissue-specific promoter, to obtain tissue specific transcription, or a regulatable promoter that can include but are not limited to hypoxia-controlled or ligand-dependent expression (e.g. tetracycline-dependent gene regulation) or sequences such as an IRES sequence to obtain translation of the messenger RNA. The transduced vector may be present in the cell stably integrated in the chromosome or as a non-integrated episomal form.

Intravascular injection includes, unless otherwise stated, intravenous and intra-

* arterial injection.

Cardiovascular means relating to the heart and the blood vessels or the circulation. Cardiovascular tissue comprises the vascular tissue of any organ, including but not limited to the vascular tissue of liver, spleen, lung, heart, brain and testis or tumor vasculature.

Gene therapy as used here mean the delivery of a gene of interest to a target cell, the introduction of said gene in said cell and the expression of said gene in a manner that it results in a therapeutic effect Lentiviral vector is any vector derived from a lentivirus, such as HIV, SIV, MW, FIV, EIAV, CAEV or BIV. Preferentially the vector is derived from HIV-1, HIV-2, SIV, FIV, EIAV or CAEV.

Animal means any animal, including human.

Cancer is any kind of cancer as known to the person skilled in the art.

Vascular diseases is any kind of vascular disease as known to the person skilled in the art and include, but are not limited to neointimal hyperplasia, myocardial and cerebral ischemia (leading to myocardial infarction and stroke, respectively), peripheral vascular disease, hypertension, pulmonary hypertension, renal artery stenosis and intracranial aneurism.

Genetic diseases include, but are not limited to cystic fibrosis and haemophilia, Alzheimer's disease, Parkinson's disease.

Brief description of the figures Figure 1: Generation of lentiviral vectors derived from HIV (human immunodeficiency virus). HIV causes progressive immunodeficiency (AIDS) which is due to the expression of several pathogenic viral proteins particularly env, tat, vif, vpu, vpr and net. None of these genes is present in the packaging cells or in the HIV-based lentiviral vector backbone. The HIV envelope could be substituted by other heterologous envelopes, such as VSV-G to generate VSV-G pseudotyped HIV-based lentiviral vectors with broad tropism. HIV vector particles were generated by transfecting 293T cells with a third-generation HIV vector plasmid. This vector backbone does not contain any HIV genes and therefore cannot express HIV viral

proteins. The necessary HIV c/s-acting elements required for packaging ( Ψ^~, A.gag),

expression, reverse transcription and integration are present in the vector. Additional c/s-acting elements such as the rev-responsive element (RRE), polypurine tract (PPT) and Woodchuck hepatitis virus post-regulatory element (PRE) were incorporated in the vector to further boost expression and vector titer. The HIV vector expresses GFP from an internal constitutive CMV promotor. The CMV/HIV LTR (R/U5) chimeric promotor is necessary to generate full-length viral genomes in the packaging cells. Only the internal C/ -promotor is active in transduced human cells, since the 3' HIVLTR, which contains an inactivating deletion (ΔLTR), replaces the chimeric CMV/R/U5 promotor upon retroviral integration. The missing viral functions of this self-inactivating vector are complemented in the packaging cells using a heterologous VSV-G expression cassette and an HIV helper construct that encodes HIV gag and pol in a conditional Rev-dependent fashion. This helper construct or the rev-expressing construct cannot be packaged in vector particles,

since the packaging sequence was deleted (ΔΨ). In addition, the helper constructs cannot be reverse transcribed or integrate in the target cells since the 3' HIV LTR was replaced with a heterologous po/yAThe resulting VSV-G pseudotyped HIV vector particles that contain the GFP transgene (HIV-GFP) are secreted by these packaging cells and can be used to transduce different target cells. Figure 2: Confocal microscopy reveals highly efficient and stable lentiviral gene transfer (2 months post-injection in neonates) in various organs including, liver, spleen, lung, heart, testis and brain, resulting in high and stable GFP reporter gene expression. Transduced pulmonary target tissue may include the pulmonary vasculature tissue and/or epithelial cells Transduced heart tissue includes cardiomyocytes and may also include vascular target cells (endothelial, smooth muscle cells). Transduced testicular tissue may include vascular target cells (endothelial, smooth muscle cells) and perhaps also cells of the intertubular tissue or other cells specific for the target tissue (25x magnification). See following figures for details on cerebral, hepatic, and splenic transduction.

Figure 3: GFP expression in brain: stable and efficient gene transfer into the vasculatur tissue brain is apparent by confocal microscopy following systemic administration of GFP-lentiviral vectors in neonates resulting in long-term (2 months), high level transgene expression. Transduced cells include vascular target cells (endothelial cells), possibly smooth muscle cells and perhaps also other cells specific for the target tissue (e.g. neurons) (magnification: left panel: 37x, right panel: 148x).

Figure 4: GFP expression in liver following systemic administration of GFP-lentiviral vectors in neonates: confocal microscopy indicates that long-term GFP expression (2 months) is predominant in hepatocytes based on their typical cuboidal morphology and tetraploidy due to binucleation (right panel) but also includes other targets particularly the liver sinusoids (arrow, left panel) and other vascular structures or cells , endothelial cells and possibly smaller Kupffer cells (right panel). This indicates highly efficient hepatic lentiviral transduction in neonates without artificial induction of hepatocyte proliferation. The hepatic transduction efficiencies (45%) are significantly higher than was has been reported previously (3-4%, Kafri et al., 1997) and can be achieved in the absence of artificial stimulation of growth stimulation in contrast to previous reports (2% without partial hepatectomy, 60% following partial hepatectomy, Park et al., 2000) (magnification: left panel: 34x, right panel: 135x).

Figure 5: GFP expression in liver following injection of lentiviral-GFP vector in 4 week old mice. See legend of figure 4 for details. The hepatic transduction efficiencies (15- 20%) are significantly higher than was has been reported previously (3-4%, Kafri et al., 1997) and can be achieved in the absence of artificial stimulation of growth stimulation in contrast to previous reports (2% without partial hepatectomy, 60% following partial hepatectomy, Park et al., 2000) (magnification: left panel: 41 x, right panel: 166x).

Figure 6: GFP expression in spleen: stable gene transfer into the spleen is highly efficient using lentiviral vectors as revealed by confocal microscopy, resulting in high levels of (GFP) transgene expression at 2 months post-injection in neonates. Transduced splenocytes include antigen-presenting cells like dendritic cells and possibly also vascular target cells (endothelial, smooth muscle cells) (magnification: left panel: 41x, right panel: 166x). Transduction of these dendritic cells may have immunomodulatory consequences and can be used for genetic vaccination (cancer, infectious diseases) or genetic tolerance induction (allergy, auto-immune diseases). Figure 7: GFP expression in liver (left panel) and spleen (right panel) following intravenous injection of lentiviral-GFP vector in 6 week old adult mice. Similar data were obtained by intraperitoneal administration (data not shown). Confocal microscopy on organs obtained 1 week post-injection indicates high levels of GFP expression in hepatocytes but also includes other targets particularly the liver sinusoids and other vascular structures or cells, endothelial cells and Kupffer cells. This indicates highly efficient hepatic lentiviral transduction in adults without artificial induction of hepatocyte proliferation. Transduced splenocytes include antigen- presenting cells like dendritic cells (and possibly also vascular target cells) (magnification: 169x).

Figure 8: Human factor IX expression in adult mice injected with lentiviral-FIX vector. Mice were injected once intravenously with 3 μg of p24 equivalent of concentrated lentiviral-FIX vector resuspended in saline with 1% bovine serum albumin (BSA). The plasma FIX levels were quantified using the Asserachrom IX: Ag ELISA kit

(Boehringer Mannheim). Pooled normal human plasma assumed to contain 5μg/ml FIX was used as standard (n= number of mice used).

Figure 9: High GFP expression in vascular target cells including human endothelial, human smooth muscle and murine fibroblastic cells transduced ex vivo with lentiviral- GFP vector (MOI=40). GFP expression could also be detected at lower MOI (MOI= 1) (data not shown). High GFP expression in saphenous vein transduced ex vivo with lentiviral-GFP vector is also shown. Examples

Example 1 : Production and characterization of lentiviral vectors.

293T cells were seeded in a cell factory and/or single tray unit (Nalge Nunc International, Roskilde, Denmark; Cat #170009A or 165250A) expanded in D10 medium (described previously, Chuah et al., 1998) and transfected when 90-100% confluent using either Gibco (Life Technologies, Merelbeke, Belgium; Cat # 18306- 019) or 5 Prime/ 3 Prime (Sanvertech, Boechout, Belgium; Cat# 2-463335) calcium phosphate transfection kit according to manufacturer's instructions. Plasmid DNA to be transfected was extracted using Qiagen Maxi or endo-free plasmid Mega kit (Westburg, Leusden, NL; Cat# 12831) according to manufacturer's instructions. Plasmid DNA was sterilized by ethanol precipitation at a concentration of 0.001 mg/ml prior to transfection. Per cell factory, the following amount of DNA was used in a total volume of 655 ml D10 medium (or 65.5 per cell tray unit): 3 mg of lentiviral-GFP vector, 1.5 mg of pMDL gag/pol RRE helper plasmid, 1.5 mg of Rev expressing plasmid, 1.5 mg pCI-VSV-G envelope-encoding plasmid, which were previously described (Dull et al., 1998; Zufferey et al., 1998). The lentiviral-GFP vector was similar to the published construct (ibid.) except that an additional polypurine tract (PPT) and post-transcriptional regulatory element from Woodchuck hepatitis virus (WPRE) had been introduced in the vector backbone to augment transgene expression and/or viral vector titer (Fig.1). A similar lentiviral vector containing the human factor IX gene ha also been constructed. The next day, medium was removed and replaced with 100 ml D10 containing 1.1 mg/ml Na-butyrate (Sigma Cat# B-5887 , Sigma, Bornem, Belgium) per cell tray. Conditioned medium was collected every 24 or 48 h-interval during the subsequent days until significant cell death and/or cell detachment occurred. Viral vector concentration was performed as, previously described after filtration with a 0.45 micrometer filter (Corning, Cat # 430770; Elscolab, Kruibeke, Belgium) at 4500 rpm overnight at 4°C with a SLA1500 rotor (VandenDriessche et al., 1999) . Titrations were performed as described previously (Chuah et a/., 1995) with minor modifications. Briefly, 2x10⁵ NIH-3T3 fibroblasts were seeded in a 6 well plate and transduced with serially diluted unconcentrated or concentrated vector-containing supernatant supplemented with polybrene or hexadimethrin bromide (Cat # 52495 Fluka, Bornem, Belgium; 8 microgram per ml final concentration). Next day, cells were washed twice with PBS and fresh D10 without polybrene was added; 48 hr post-transfection, number of fluorescent cells per microscopic field (at least 3-10 fields) were counted on which basis the total number of fluorescent cells per ml viral vector-containing supernatant could be calculated (= titer in TU/ml, transducing units per ml). Alternatively, titer of the lentiviral-FIX vector was determined by quantifying p24 levels by ELISA as described previously (Naldini et al., 1996).

Example 2: In vivo transduction

Newborn immunodeficient mice (1-2 days old FVIII KO-SCID; VandenDriessche etal., 1999) were injected intravenously as described (ibid.) with a total of 0.2 ml of concentrated lentiviral-GFP vector (titer 1 ,9x10⁸ tu/ml; thus total vector dose: 4x10⁷ tu) supplemented with 0.040mg/ml polybrene over 2 consecutive days. Mice were dissected about 2 months post-injection. Four week old FVIII KO-SCID mice were injected intravenously by tail-vein injection with a total of 0.2 ml of concentrated lentiviral-GFP vector (titer 3.3x10⁸ tu/ml; thus total vector dose: 7x10⁷ tu). Six-week old adult FVIII KO-SCID mice were injected intravenously by tail-vein injection with a total of 0.5 ml of concentrated lentiviral-GFP vector (titer 10⁸ tu/ml; thus total vector dose: 5x10⁷ tu). Different organs were subjected to confocal microscopical analysis according to standard procedures and using software supplied by manufacturer (Zeiss Axiovert 100 M / Leica). The results are shown in figs. 2-7. These data demonstrate that systemic administration of lentiviral vectors by intravenous injection into neonatal animals lead to efficient gene transfer into various organs and paves the way for an approach whereby upon vector envelope modification targetable, cell- specific lentiviral vectors can be employed.

In addition to the use of GFP as reporter protein, a secretable protein such as human coagulation factor IX (FIX) was used. Adult SCID mice were injected once intravenously with 3 μg of p24 equivalent of concentrated lentiviral-FIX vector

resuspended in saline with 1% bovine serum albumin (BSA). The plasma FIX levels were monitored weekly. Blood taken from the tail veins was collected into tubes containing 0.1 M Na-citrate (10% v/v). Plasma was obtained by centrifugation at 1000xg for 10 min and subsequently stored at -20°C. The concentration of human FIX was determined using the Asserachrom IX:Ag ELISA kit (Boehringer Mannheim). Plasma samples were diluted 1 :3 with the buffer supplied in the kit. Pooled normal

human plasma assumed to contain 5μg/ml FIX was used as standard either diluted in the sample diluent or in mouse serum. The results are shown in fig. 8 (n= number of mice used). Adult FVIII-deficient hemophilic mice were injected intravenously with concentrated lentiviral-FVIII vector. Injected vector dose corresponds to 1-4x10 (9) transducing units as determined by p24 ELISA using HIV-CMV-GFP with known functional titer as reference. Blood taken from the tail veins was collected into tubes containing 0.11VI Na-citrate (10% v/v). Plasma was obtained by centrifugation at 1000xg for 10 min and subsequently stored at -20°C. The concentration of human FVIII was determined using a functional FVIII COATEST kit (Chromogenix). Purified human FVIII was used as standard diluted in mouse plasma. The results show that lentiviral transduction of human coagulation factor VIII resulted in therapeutic levels of FVIII (10 ng/ml) in hemophilic mice. To our knowledge, it has never been shown that systemic administration of lentiviral vectors can result in therapeutic FVIII levels.

Example 3: Ex vivo transduction

Human umbilical vein endothelial cells and primary human smooth muscle cells (10⁵ cells/ml) were transduced overnight with concentrated HIV-GFP vector (MOI=40) supplemented with polybrene (Cat # 52495 Fluka, Bornem, Belgium; 8 microgram per ml final concentration). GFP expression was monitored 48 hr later. Similarly, porcine saphenous vein was cut in 2-3 mm slices and exposed to 0.1 ml of concentrated lentiviral-GFP vector (titer: 2.5x10⁹ tu/ml; total vector dose: 2.5x10⁸tu) in the presence of 0.3 ml standard RPMI 1640 medium supplemented with 2 g/l sodium bicarbonate, penicillin/streptomycin (100 U/ml) + 0.8 mM L-glutamine (Life Technologies, Merelbeke, Belgium). The next day, slices were washed with PBS, and standard RPMI 1640 medium was added suplemented with 30% fetal bovine serum. The sections were embedded in Tissue-Tek, frozen in liquid nitrogen, fixed with 4% paraformaldehyde and cryosectioned according to standard protocol (5-15 μm). GFP

expression was monitored by fluorescent microscopy. The results are shown in fig. 9.

References

Blomer, U., Naldini, L, Kafri, T., Trono, D., Verma, I. M. and Gage, F. H. (1997). Highly efficient and sustained gene transfer in adult neurones with a lentivirus vector. J Virol 71.- 6641-6649.

Chuah M.K., Vandendriessche T. and Morgan R.A. (1995). Development and analysis of retroviral vectors expressing human factor VIII as a potential gene therapy for hemophilia A. Hum Gene Ther, 6, 1363-1377.

Chuah M.K., Bre s H., Vanslembrouck V., Collen D. and Vandendriessche T. (1998). Bone marrow stromal cells as targets for gene therapy of hemophilia A. Hum Gene Ther 9:353-365.

Dull T., Zufferey R., Kelly M., Mandel R.J., Nguyen M., Trono D. and Naldini L. (1998). A third-generation lentivirus vector with a conditional packaging system. J Virol, 72:8463-8471.

Han, J.J., Mhatre, A.N., Wareing, M., Pettis, R., Gao, W.Q., Zufferey, R.N., Trono, R.N. and Lalwani, A.K. (1999). Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector. Hum Gen Tiber 10: 1867-1873.

Kafri, T., Blomer, U., Peterson, D. A., Gage, F. H. and Verma, I. M. (1997). Sustained expression of genes delivered into liver and muscle by lentiviral vectors. Nature Genetics 17: 314-317.

Lewis PF, Emerman M (1994). Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol 68:510-516. Miyoshi, H., Takahashi, M., Gage, F. H. and Verma, I. M. (1997). Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc NatAcad Sci USA 94: 10319-10323.

Naldini, L., Blomer, U., Gage, F., Trono, D., and Verma, I. Efficient transfer, integration, and sustained long-trem expression of the transgene in adult rat brains injected with a lentiviral vector. Proc NatAcad Sci USA 93: 11382-11388.

Park F., Ohashi K., Chiu W., Naldini L. and Kay M.A. (2000). Efficient lentiviral transduction of liver requires cell cycling in vivo. Nat Genet, 24: 49-52.

VandenDriessche T., Vanslembrouck V., Goovaerts I., Zwinnen H., Vanderhaeghen M.L., Collen D. and Chuah M.K. (1999). Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VI I l-def icient mice. Proc Natl Acad Sci U SA, 1999, 96: 10379- 10384.

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Claims

1. Method to obtain systemic delivery and/or stable systemic transduction of a lentiviral vector, comprising introduction of said lentiviral vector into an animal.

2. Method according to claim 1 where said introduction is an intravascular injection.

3. Method according to claim 1 where said introduction is an intravenous injection

4. Method according to claim 1-3, where said animal is a neonatal animal.

5. Method according to claim 1-4, wherein said vector is a VSV-G pseudotyped HIV derived lentiviral vector.

6. Method according to claim 1-5 in which the transduction is in the cardiovascular tissue.

7. Method according to claim 6 in which the transduction is in the endothelial cells and/or the smooth muscle cells of the cardiovascular tissue.

8. Method according to claim 6 or 7 in which the transduction is in the vascular tissue of the liver, spleen, lung, heart, brain and/or testis. 9. Method to obtain stable transduction of a lentiviral vector into the cardiovascular system, comprising introduction of said lentiviral vector into an animal.

10. Method according to claim 9 where said introduction is an intravascular injection.

11. Method according to claim 9, in which said introduction comprises introduction of said lentiviral vector into vascular tissue obtained from said animal. 12. Method according to claim 9-11, wherein said vector is a VSV-G pseudotyped HIV derived lentiviral vector.

13. Use of a method according to any of claims 1-12 for performing gene therapy.

14. Use of a method according to claim 13 to treat vascular diseases.

15. Use of a method according to claim 13 to treat cancer.

6. Use of a method according to claim 13 to treat genetic diseases.