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  Pharmaceutical Patents  

 

Title:  Methods of treating lysosomal storage related diseases by gene therapy
United States Patent: 
7,592,321
Issued: 
September 22, 2009

Inventors:
 Whitley; Chester B. (Brooklyn Park, MN), McIvor; R. Scott (St. Louis Park, MN)
Appl. No.:
 11/057,410
Filed:
 February 14, 2005


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

Isolated nucleic acid-based vectors and lentivirus vectors, and methods of using those vectors to inhibit or prevent metabolic disorders in a mammal, are provided.

Description of the Invention

SUMMARY OF THE INVENTION

The invention provides a lentivirus vector comprising a nucleic acid segment encoding a gene product such as a protein, the absence or reduced levels of which are associated with a disorder in a mammal, a disorder including, but not limited to, a metabolic disorder, e.g., a lysosomal storage disease, hemophilia, or adrenoleukodystrophy. A lentivirus includes ovine, caprine, equine, bovine and primate, e.g., HIV-1, HIV-2 and SIV, lentiviruses. Also provided is a method in which a recombinant lentivirus comprising a nucleic acid segment encoding a gene product, the absence or reduced levels of which in a mammal are associated with a disorder, is administered to a mammal having or at risk of having such a disorder, in an amount effective to prevent, inhibit or treat at least one symptom associated with the disorder, e.g., a neurological symptom. In one embodiment, the recombinant virus is administered into a vascular compartment, e.g., intravenously, of the mammal. Preferred amounts of virus include, but are not limited to, 1.times.10.sup.3 to 1.times.10.sup.15 TU, e.g., 1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7, 1.times.10.sup.8, 1.times.10.sup.9, 1.times.10.sup.10, 1.times.10.sup.11, 1.times.10.sup.12, 1.times.10.sup.13, 1.times.10.sup.14 or 1.times.10.sup.15 TU, although other amounts may be efficacious. Preferably, the mammal is a neonate or juvenile, although it is envisioned that adult mammals, and the developing embryo or fetus in utero, may also be treated.

In one embodiment, the recombinant lentivirus encodes a lysosomal enzyme and is administered in an amount which is effective to prevent, inhibit or treat a lysosomal storage disease in a mammal. Lysosomal storage diseases include, but are not limited to, mucopolysaccharidosis diseases, for instance, mucopolysaccharidosis type I, e.g., Hurler syndrome and the variants Scheie syndrome and Hurler-Scheie syndrome (a deficiency in alpha-L-iduronidase); Hunter syndrome (a deficiency of iduronate-2-sulfatase); mucopolysaccharidosis type III, e.g., Sanfilippo syndrome (A, B, C or D; a deficiency of heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminide N-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IV e.g., mucopolysaccharidosis type IV, e.g., Morquio syndrome (a deficiency of galactosamine-6-sulfate sulfatase or beta-galactosidase); mucopolysaccharidosis type VI. e.g., Maroteaux-Lamy syndrome (a deficiency of arylsulfatase B); mucopolysaccharidosis type II; mucopolysaccharidosis type III (A, B, C or D; a deficiency of heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminide N-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IV (A or B; a deficiency of galactosamine-6-sulfatase and beta-galatacosidase); mucopolysaccharidosis type VI (a deficiency of arylsulfatase B); mucopolysaccharidosis type VII (a deficiency in beta-glucuronidase); mucopolysaccharidosis type VIII (a deficiency of glucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IX (a deficiency of hyaluronidase); Tay-Sachs disease (a deficiency in alpha subunit of beta-hexosaminidase); Sandhoff disease (a deficiency in both alpha and beta subunit of beta-hexosaminidase); GM1 gangliosidosis (type I or type II); Fabry disease (a deficiency in alpha galactosidase); metachromatic leukodystrophy (a deficiency of aryl sulfatase A); Pompe disease (a deficiency of acid maltase); fucosidosis (a deficiency of fucosidase); alpha-mannosidosis (a deficiency of alpha-mannosidase); beta-mannosidosis (a deficiency of beta-mannosidase), ceroid lipofuscinosis, and Gaucher disease (types I, II and III; a deficiency in glucocerebrosidase), as well as disorders such as Hermansky-Pudlak syndrome; Amaurotic idiocy; Tangier disease; aspartylglucosaminuria; congenital disorder of glycosylation, type Ia; Chediak-Higashi syndrome; macular dystrophy, corneal, 1; cystinosis, nephropathic; Fanconi-Bickel syndrome; Farber lipogranulomatosis; fibromatosis; geleophysic dysplasia; glycogen storage disease I; glycogen storage disease Ib; glycogen storage disease Ic; glycogen storage disease III; glycogen storage disease IV; glycogen storage disease V; glycogen storage disease VI; glycogen storage disease VII; glycogen storage disease 0; immunoosseous dysplasia, Schimke type; lipidosis; lipase b; mucolipidosis II; mucolipidosis II, including the variant form; mucolipidosis IV; neuraminidase deficiency with beta-galactosidase deficiency; mucolipidosis I; Niemann-Pick disease (a deficiency of sphingomyelinase); Niemann-Pick disease without sphingomyelinase deficiency (a deficiency of a npc1 gene encoding a cholesterol metabolizing enzyme); Refsum disease; Sea-blue histiocyte disease; infantile sialic acid storage disorder; sialuria; multiple sulfatase deficiency; triglyceride storage disease with impaired long-chain fatty acid oxidation; Winchester disease; Wolman disease (a deficiency of cholesterol ester hydrolase); Deoxyribonuclease I-like 1 disorder, arylsulfatase E disorder; ATPase, H+ transporting, lysosomal, subunit 1 disorder; glycogen storage disease IIb; Ras-associated protein rab9 disorder; chondrodysplasia punctata 1, X-linked recessive disorder; glycogen storage disease VIII; lysosome-associated membrane protein 2 disorder; Menkes syndrome; congenital disorder of glycosylation, type Ic; and sialuria. In particular, the invention is useful to prevent, inhibit or treat lysosomal storage diseases wherein the lysosomal enzyme is trafficked to the lysosome (within the cell and between cells) by specific glycosylation. For most lysosomal enzymes and their corresponding diseases, this would be by means of a terminal mannose-6-phosphate, however, it also includes terminal mannose glycosylation, e.g., in the case of beta-glucocerebrosidase deficiency responsible for Gaucher disease. Thus, in one embodiment, the lentivirus vector of the invention is useful to prevent, inhibit or treat lysosomal storage diseases including but are not limited to, mucopolysaccharidosis diseases, for instance, mucopolysaccharidosis type I, e.g., Hurler syndrome and the variants Scheie syndrome and Hurler-Scheie syndrome (a deficiency in alpha-L-iduronidase); mucopolysaccharidosis type II, e.g., Hunter syndrome (a deficiency of iduronate-2-sulfatase); mucopolysaccharidosis type III, e.g., Sanfilippo syndrome (A, B, C or D; a deficiency of heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminide N-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IV, e., Morquio syndrome (a deficiency of galactosamine-6-sulfate sulfatase or beta-galactosidase); mucopolysaccharidosis type VI, e.g., Maroteaux-Lamy syndrome (deficiency of arylsulfatase B); mucopolysaccharidosis type VII, e.g., Sly syndrome (a deficiency in beta-glucuronidase); Tay-Sachs disease (a deficiency in alpha subunit of beta-hexosaminidase); Sandhoff disease (a deficiency in both alpha and beta subunit of beta-hexosaminidase); GM1 gangliosidosis; Fabry disease (a deficiency in alpha-galactosidase); metachromatic leukodystrophy (a deficiency of aryl sulfatase A); Pompe disease (a deficiency of acid maltase); fucosidosis (a deficiency of fucosidase); alpha-mannosidosis (a deficiency of alpha-mannosidase); beta-mannosidosis (a deficiency of beta-mannosidase), ceroid lipofuscinosis, and Gaucher disease (types I, II and III; a deficiency in glucocerebrosidase). As described herein, a single administration of a lentivirus encoding alpha-L-iduronidase to newborn Hurler syndrome mice resulted in normal patterns of behavior for the treated mice relative to untreated mice. Administration of the lentivirus to newborns likely resulted in an increased efficiency of transduction which may in turn be due to the presence of cells that are more susceptible to infection in the newborn. Early therapy, e.g., prenatal or in newborns, for metabolic disorders such as lysosomal storage diseases may thus be particularly efficacious.

In one embodiment, a recombinant lentivirus encoding a lysosomal enzyme is administered to a mammal in an amount which is effective to increase the level and/or activity of one or more lysosomal storage proteins, e.g., enzymes, and/or decrease skeletal deformity including kyphoscoliosis, scoliosis, deformity or arthritis of the hip joints, contractures of the digits or larger joints at the knees, ankles, elbows and shoulders, disfigurement of the face, recurrent and chronic ear infections, enlargement, dysfunction of an organ such as the liver, spleen or heart, obstuction of the coronary arteries causing myocardial infarction, respiratory abnormality such as obstructive airway disease, reactive airway disease or pneumonia, brain or other nervous system damage, and/or dysfunction such as hydrocephalus, cranial nerve compression, hearing loss, blindness, spinal cord compression.

In one embodiment, a recombinant lentivirus encoding a lysosomal enzyme is administered to a mammal in an amount which is effective to increase longevity, preserve intellect, e.g., measured by intelligence quotient (IQ), reduce ear infections, reduce skeletal deformity with improved ambulation, e.g., measured in a 6-minute walk test or other measurements of endurance, reduce organ size (e.g., liver, spleen), improve respiratory function, e.g., measured by improved by spirometry, normalize of organ cellular architecture, e.g., observed by decreased pathology (reduced lyosomal vacuolization or other microscopic pathology), decrease occlusion of the coronary arteries, reduce aberrant thickening of the meninges of the central nervous system, prevent or reduce hydrocephalus of the brain, decrease levels of pathologic substrates such as decreased glycosaminoglycan in the liver and other tissues, urine, or cerebrospinal fluid, and/or increase levels of a deficient enzyme such as alpha-L-iduronidase in liver tissue, white blood cells or plasma.

In one embodiment, a recombinant lentivirus encoding a lysosomal enzyme is administered intravenously to a mammal, e.g., a fetus (prenatal delivery), an infant (e.g., a human from birth to 2 years of age), a child (e.g., a human from over 2 years to 12 years or age), a juvenile (e.g., a human from over 12 years to 18 years of age), or an adult (e.g., a human older than 18 years of age).

In another embodiment, the invention provides a lentivirus vector comprising a nucleic acid sequence encoding a clotting factor, e.g., Factor VIII or Factor IX, and a method to prevent, inhibit or treat a mammal having or at risk of having the clotting disorder which employs a recombinant lentivirus comprising the vector. Preferably, the recombinant lentivirus is administered to a vascular compartment of the mammal.

Further provided is a lentivirus vector comprising a nucleic acid sequence encoding an ABC protein such as a peroxisomal transport protein, e.g., the X-ALD protein (ALDP), the adrenoleukodystrophy-related protein (ALDRP), the 70 kDa peroxisomal membrane protein (PMP70), or a PMP70-related protein, and a method to prevent, inhibit or treat a mammal having or at risk of an adrenoleukodystrophy which employs a recombinant lentivirus comprising the vector. Preferably, the lentivirus is administered to a vascular compartment of the mammal.

The invention also provides a recombinant lentivirus of the invention, a host cell transfected with a lentivirus vector of the invention, e.g., eukaryotic host cells including mammalian host cells such as human, non-human primate, canine, caprine, feline, bovine, equine, swine, ovine, rabbit or rodent cells, a host cell infected, e.g., ex vivo, with a recombinant lentivirus of the invention, and a method of expressing a biologically active protein in a cell which employs a lentivirus vector or lentivirus of the invention which encodes the biologically active protein. A "biologically active" protein is a protein which has substantially the same activity, e.g., at least 80%, more preferably at least 90%, the activity of a corresponding wild-type (functional) protein.

Also provided is a kit comprising a recombinant lentivirus of the invention, e.g., a lyophilized or frozen preparation of recombinant lentivirus.

Mucopolysaccharidosis (MPS) type VII is an autosomal recessive lysosomal storage disease resulting from deficiency of beta-glucuronidase due to mutations of the corresponding gene for beta-glucuronidase, GUSB. As described herein, a plasmid was constructed to express the human GUSB cDNA under the transcriptional regulation of a hybrid promoter-enhancer (CAGGS) containing the chicken beta-actin enhancer and CMV early promoter. Sleeping Beauty transposon IR sequences were included to examine the potential for integration into the cell chromosome. This transposed plasmid pT-CAGGS-GUSB was administered by hydrodynamic injection (i.e., intravenous infusion in a volume equal to 10% of body weight, over about 8-10 seconds) into the tail vein of mice ranging from 4 to 23 weeks of age. The pT-CAGGS-GUSB plasmid was administered (25 mcg/animal) alone (Group 1), or with transposase plasmid pSBI0, at a transposon:transposase molar ratio of 1:1 (Group 2), or 10:1 (Group 3). Forty-eight hours after injection, plasma beta-glucuronidase enzymatic activity in treated MPS mice was markedly elevated (1,552-7,711 mmol/ml/hr, n=14) in comparison to that of wild-type, untreated or sham-treated mice (9-15 nmol/ml/hr, n=6). In liver, beta-glucuronidase activity in treated MPS mice was also markedly elevated (1,860-6,185 nmol/mg/hr, n=4) compared to normal levels (86-188 nmol/mg/hr). Notably, the liver tissue of MPS mice receiving pTCAGGS-GUSB stained uniformly positive for beta-glucuronidase activity, including both Kupffer cells and hepatocytes. One week after injection, plasma beta-glucuronidase activity was reduced relative to day 2 levels: Group 1, 59-93% of the 2-day levels; Group 2, 21-36%; and Group 3, 33-63% (n=4 in each group). Beta-glucuronidase levels in the liver and spleen were 184-185 nmol/mg/hr and 4,534-6,080, respectively, while levels in other organs were lower (heart 94-98, lung 49-65, kidney 59, and undetectable in the brain). Two months after injection, beta-glucuronidase activity remained at therapeutic levels in animals receiving pT-CAGGS-GUSB plasmid alone. Histochemical studies showed staining for beta-glucuronidase activity throughout the liver and spleen. Remarkably, mice co-injected with pSBIO had much lower levels of beta-glucuronidase activity. Morphometric analysis of inclusion morphology demonstrated that clearing of hepatic lysosomal pathology was related to the level of beta-glucuronidase, and that mice receiving pT-CAGGS-GUSB plasmid alone were completely clear of pathology.

Thus, hydrodynamic infusion of the pT-CAGGS-GUSB transposon delivered DNA to liver with marked increase in enzyme activity, with the highest levels in blood ever achieved. GUSB enzymatic activity was d throughout the liver transiently reaching levels 10- to 1,000-fold of normal levels, levels which are above those that would be curative in newborns.

The results described herein with the lentivirus and plasmid vectors of the invention were surprising as the intravenous administration of other vectors did not show the extent of correction observed with the lentivirus and plasmid vectors. Moreover, PCR analysis of gonads, e.g., testes, showed virtually no evidence of viral vector sequences, indicating a decreased risk for germ line transmission. Further, viral vector sequences were surprisingly detected in bone marrow stem cells after intravenous administration of a lentivirus vector of the invention to a mammal and so those vectors are particularly useful for systemic expression of therapeutic genes.

The invention provides a method to prevent, inhibit or treat a metabolic disorder in a mammal via the hydrodynamic infusion of a plasmid encoding a gene product, the expression of which in the mammal prevents, inhibits, or treats one or more symptoms of the disorder. In one embodiment, a fetus or neonate is infused via the umbilical cord with a vector of the invention.

The invention provides a method to prevent, inhibit or treat a metabolic disorder such as one characterized by the absence or reduced levels of a lysosomal protein in a mammal. The method comprises administering to a mammal, e.g., to a vascular compartment of a mammal having or at risk of the disorder an effective amount of an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the protein, e.g., a biologically active protein. In one embodiment, the nucleic acid molecule comprises a promoter operably linked to the nucleic acid sequence.

The invention also provides isolated nucleic acid-based vectors to inhibit or treat metabolic disorders, e.g., lysosomal storage disease such as mucopolysaccharidosis type I diseases, e.g., Hurler syndrome, mucopolysaccharidosis type II diseases, e.g., Hunter syndrome, mucopolysaccharidosis type II diseases, e.g., Sanfilippo syndrome, mucopolysaccharidoses type VII diseases, e.g., Sly disease, Fabry disease, Gaucher disease as well as hemophilia, e.g., Factor VIII or factor IX deficiency. Further provided is a method to prevent, inhibit or treat a metabolic disorder in a mammal which employs an isolated nucleic acid vector of the invention, e.g., in an amount effective to prevent, inhibit or treat at least one symptom associated with the disorder, e.g., a neurological symptom associated with the disorder. In one embodiment, the mammal is an adult. In one embodiment, the isolated nucleic acid vector of the invention is administered two or more times to the mammal.

In another embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a clotting factor, e.g., Factor VM or Factor IX, and a method to prevent, inhibit or treat a mammal having or at risk of having the clotting disorder which employs a vector comprising the nucleic acid molecule. Preferably, the vector is administered to a vascular compartment of the mammal.

Further provided is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an ABC protein such as a peroxisomal transport protein, e.g., the X-ALD protein (ALDP), the adrenoleukodystrophy-related protein (ALDRP), the 70 kDa peroxisomal membrane protein (PMP70), or a PMP70-related protein, and a method to prevent, inhibit or treat a mammal having or at risk of an adrenoleukodystrophy which employs a vector comprising the nucleic acid molecule. Preferably, the lentivirus is administered to a vascular compartment of the mammal.

The invention also provides an isolated nucleic acid molecule of the invention, a host cell transfected with the isolated nucleic acid molecule of the invention, e.g., eukaryotic host cells including mammalian host cells such as human, non-human primate, canine, caprine, feline, bovine, equine, swine, ovine, rabbit or rodent cells, a host cell transfected, e.g., ex vivo, with an isolated nucleic acid molecule of the invention, and a method of expressing a biologically active protein in a cell which employs a vector comprising a nucleic acid molecule of the invention which encodes the biologically active protein.

DETAILED DESCRIPTION OF THE INVENTION

Vectors for Recombinant Lentivirus Production

The lentiviral genome and the proviral DNA have the three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), a protease and an integrase; and the env gene encodes viral envelope glycoproteins. The 5' and 3`LTR`s serve to promote transcription and polyadenylation of the virion RNA's. The LTR contains all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif vpr, tat, rev, vpu, nef and vpx (in HIV-1, HIV-2 and/or SIV).

Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins.

The invention provides a method of producing a recombinant lentivirus capable of infecting a cell, e.g., non-dividing cell, comprising transfecting a suitable host cell with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat. As will be disclosed hereinbelow, vectors lacking a functional tat gene are desirable for certain applications. Thus, for example, a first vector can provide a nucleic acid encoding a viral gag and a viral pol and another vector can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene, herein identified as a transfer vector, into that packaging cell yields a producer cell which releases infectious viral particles carrying the heterologous gene of interest.

Generally the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest.

According to the above-indicated configuration of vectors and heterologous genes, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from any virus, including retroviruses, e.g., lentiviruses, and heterologous viruses such as VSV. The env preferably is an envelope protein which allows transduction of cells of human and other species.

It may be desirable to target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type. By inserting a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific. Retroviral vectors can be made target-specific by inserting, for example, a glycolipid or a protein. Targeting often is accomplished by using an antigen-binding portion of an antibody or a recombinant antibody-type molecule, such as a single chain antibody, to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific methods to achieve delivery of a retroviral vector to a specific target.

Examples of retroviral-derived env genes include, but are not limited to: Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), murine mammary tumor virus (4 uMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV), Rous sarcoma virus (RSV), and env genes of amphotropic viruses. Other env genes such as Vesicular stomatitis virus (VSV) protein G (VSV G), that of hepatitis viruses and of influenza also can be used.

The vector providing the viral env nucleic acid sequence is associated operably with regulatory sequences, e.g., a promoter or enhancer. The regulatory sequence can be any eukaryotic promoter or enhancer, including for example, the Moloney murine leukemia virus promoter-enhancer element, the human cytomegalovirus (CMV) enhancer or the vaccinia P7.5 promoter. In some cases, such as the Moloney murine leukemia virus promoter-enhancer element, the promoter-enhancer elements are located within or adjacent to the LTR sequences.

Preferably, the regulatory sequence is one which is not endogenous, i.e., it is heterologous, to the lentivirus from which the vector is being constructed. Thus, if the vector is being made from SIV, the SIV regulatory sequence found in the SIV LTR would be replaced by a regulatory element which does not originate from SIV.

While VSV G protein is a desirable env gene because VSV G confers broad host range on the recombinant virus, VSV G can be deleterious to the host cell. Thus, when a gene such as that for VSV G is used, it is preferred to employ an inducible promoter system so that VSV G expression can be regulated to minimize host toxicity when VSV G is expression is not required. For example, the tetracycline-regulatable gene expression system of Gosse et al. (1992) can be employed to provide for inducible expression of VSV G when tetracycline is withdrawn from the transfected cell. Thus, the tet/VP16 transactivator is present on a first vector and the VSV G coding sequence is cloned downstream from a promoter controlled by tet operator sequences on another vector.

The heterologous nucleic acid sequence of interest, the transgene, is linked operably to a regulatory nucleic acid sequence. As used herein, the term "heterologous" nucleic acid sequence refers to a sequence that originates from a foreign species, or, if from the same species, it may be substantially modified from the original form. Alternatively, an unchanged nucleic acid sequence that is not expressed normally in a cell is a heterologous nucleic acid sequence.

The term "operably linked" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. Preferably, the heterologous sequence is linked to a promoter, resulting in a chimeric gene. The heterologous nucleic acid sequence is preferably under control of either the viral LTR promoter-enhancer signals or of an internal promoter, and retained signals within the retroviral LTR can still bring about efficient expression of the transgene.

The heterologous gene of interest can be any nucleic acid of interest which can be transcribed. Generally the foreign gene encodes a polypeptide. Preferably the polypeptide has some therapeutic benefit. The polypeptide may supplement deficient or nonexistent expression of an endogenous protein in a host cell. The polypeptide can confer new properties on the host cell, such as a chimeric signalling receptor, see U.S. Pat. No. 5,359,046. The artisan can determine the appropriateness of a heterologous gene practicing techniques taught herein and known in the art. For example, the artisan would know whether a heterologous gene is of a suitable size for encapsidation and whether the heterologous gene product is expressed properly.

The method of the invention may also be useful for neuronal, glial, fibroblast or mesenchymal cell transplantation, or "grafting", which involves transplantation of cells infected with the recombinant lentivirus of the invention ex vivo, or infection in vivo into the central nervous system or into the ventricular cavities or subdurally onto the surface of a host brain. Such methods for grafting will be known to those skilled in the art and are described in Neural Grafting in the Mammalian CNS, Bjorklund & Stenevi, eds. (1985).

For diseases due to deficiency of a protein product, gene transfer could introduce a normal gene into the affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations. For example, it may be desirable to insert a Factor VIII or IX encoding nucleic acid into a lentivirus for infection of a muscle, spleen or liver cell.

The promoter sequence may be homologous or heterologous to the desired gene sequence. A wide range of promoters may be utilized, including a viral or a mammalian promoter. Cell or tissue specific promoters can be utilized to target expression of gene sequences in specific cell populations. Suitable mammalian and viral promoters for the instant invention are available in the art.

Optionally during the cloning stage, the nucleic acid construct referred to as the transfer vector, having the packaging signal and the heterologous cloning site, also contains a selectable marker gene. Marker genes are utilized to assay for the presence of the vector, and thus, to confirm infection and integration. The presence of a marker gene ensures the selection and growth of only those host cells which express the inserts. Typical selection genes encode proteins that confer resistance to antibiotics and other toxic substances, e.g., histidinol, puromycin, hygromycin, neomycin, methotrexate etc. and cell surface markers.

The recombinant virus of the invention is capable of transferring a nucleic acid sequence into a mammalian cell. The term, "nucleic acid sequence", refers to any nucleic acid molecule, preferably DNA, as discussed in detail herein. The nucleic acid molecule may be derived from a variety of sources, including DNA, cDNA, synthetic DNA, RNA or combinations thereof. Such nucleic acid sequences may comprise genomic DNA which may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with promoter regions, poly A sequences or other associated sequences. Genomic DNA may be extracted and purified from suitable cells by means well known in the art. Alternatively, messenger RNA (mRNA) can be isolated from cells and used to produce cDNA by reverse transcription or other means.

Preferably, the recombinant lentivirus produced by the method of the invention is a derivative of human immunodeficiency virus (HIV). The env will be derived from a virus other than HIV.

Thus, three or more vectors, e.g., in one or more plasmids, which provide all of the functions required for packaging of recombinant virions, such as, gag, pol, env, tat and rev, can be employed to prepare recombinant lentivirus. As noted herein, tat may be deleted. There is no limitation on the number of vectors which are utilized so long as the vectors are used to transform and to produce the packaging cell line to yield recombinant lentivirus.

The vectors are introduced via transfection or infection into the packaging cell line. The packaging cell line produces viral particles that contain the vector genome. Methods for transfection or infection are well known by those of skill in the art. After cotransfection of the packaging vectors and the transfer vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art.

Thus, the packaging constructs can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker encoding, for example, neomycin resistance, DHFR, Gln synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. The selectable marker gene can be linked physically to the packaging genes in the construct.

Stable cell lines wherein the packaging functions are configured to be expressed by a suitable packaging cell are known. For example, see U.S. Pat. No. 5,686,279; and Ory et al. (1996), which describe packaging cells.

Zufferey et al. (1997) teach a lentiviral packaging plasmid wherein sequences 3' of pol including the HIV-1 env gene are deleted. The construct contains tat and rev sequences and the 3' LTR is replaced with poly A sequences. The 5' LTR and psi sequences are replaced by another promoter, such as one which is inducible. For example, a CMV promoter or derivative thereof can be used.

The packaging vectors of interest may contain additional changes to the packaging functions to enhance lentiviral protein expression and to enhance safety. For example, all of the HIV sequences upstream of gag can be removed. Also, sequences downstream of env can be removed. Moreover, steps can be taken to modify the vector to enhance the splicing and translation of the RNA.

To provide a vector with an even more remote possibility of generating replication competent lentivirus, lentivirus packaging plasmids wherein tat sequences, a regulating protein which promotes viral expression through a transcriptional mechanism, are deleted functionally. Thus, the tat gene can be deleted, in part or in whole, or various point mutations or other mutations can be made to the tat sequence to render the gene non-functional. An artisan can practice known techniques to render the tat gene non-functional.

The techniques used to construct vectors, and to transfect and to infect cells, are practiced widely in the art. Practitioners are familiar with the standard resource materials which describe specific conditions and procedures.

A lentiviral packaging vector is made to contain a promoter and other optional or requisite regulatory sequences as determined by the artisan, gag, pol rev, env or a combination thereof, and with specific functional or actual excision of tat, and optionally other lentiviral accessory genes.

Lentiviral transfer vectors (Naldini et al., 1996) have been used to infect human cells growth-arrested in vitro and to transduce neurons after direct injection into the brain of adult rats. The vector was efficient at transferring marker genes in vivo into the neurons and long term expression in the absence of detectable pathology was achieved. Another version of the lentiviral vector in which the HIV virulence genes env, vif, vpr; vpu and nef were deleted without compromising the ability of the vector to transduce non-dividing cells, represents a substantial improvement in the biosafety of the vector (Zufferey et al., 1997).

In transduced cells, the integrated lentiviral vector generally has an LTR at each termini. The 5' LTR may cause accumulation of "viral" transcripts that may be the substrate of recombination, in particular in HIV-infected-cells. The 3' LTR may promote downstream transcription with the consequent risk of activating a cellular protooncogene. The U3 sequences comprise the majority of the HIV LTR. The U3 region contains the enhancer and promoter elements that modulate basal and induced expression of the HIV genome in infected cells and in response to cell activation. Several of the promoter elements are essential for viral replication. Some of the enhancer elements are highly conserved among viral isolates and have been implicated as critical virulence factors in viral pathogenesis. The enhancer elements may act to influence replication rates in the different cellular target of the virus (Marthas et al., 1993). Also, enhancers in either LTR can activate transcription of neighboring genes.

As viral transcription starts at the 3' end of the U3 region of the 5' LTR, this U3 region (including the promoter and enhancer) is not included in the viral mRNA, and a copy thereof from the 3' LTR acts as template for the generation of the U3 region of both LTR's in the subsequently integrated provirus. If the U3 region of the 3' LTR is altered in a retroviral vector construct so as to eliminate the promoter and enhancer, the vector RNA still is produced from the intact 5' LTR in producer cells, but cannot be regenerated in target cells. Transduction of such a vector results in the transcriptional inactivation of both LTR's in the progeny virus. Thus, the retrovirus is self-inactivating (SIN) and those vectors are known as Sin transfer vectors.

There are, however, limits to the extent of the deletion at the 3' LTR. First, the 5' end of the U3 region serves another essential function in vector transfer, being required for integration (terminal dinucleotide+att sequence). Thus, the terminal dinucleotide and the att sequence may represent the 5' boundary of the U3 sequences which can be deleted. In addition, some loosely defined regions may influence the activity of the downstream polyadenylation site in the R region. Excessive deletion of U3 sequence from the 3' LTR may decrease polyadenylation of vector transcripts with adverse consequences both on the titer of the vector in producer cells and the transgene expression in target cells. On the other hand, limited deletions may not abrogate the transcriptional activity of the LTR in transduced cells.

U3 deletions in a HIV LTR can span from nucleotide -418 of the U3 LTR to the indicated position: SIN-78, SIN-45, SIN-36 and SIN-18. Lentiviral vectors with almost complete deletion of the U3 sequences from the 3' LTR were developed without compromising either the titer of vector in producer cells or transgene expression in target cells. The most extensive deletion (-418 to -18) extends as far as to the TATA box, therefore abrogating any transcriptional activity of the LTR in transduced cells. Thus, the lower limit of the 3' deletion may extend as far as including the TATA box. The deletion may be of the remainder of the U3 region up to the R region. Surprisingly, the average expression level of the transgene is higher in cells transduced by the SIN vectors as compared to more intact vectors.

The 5' LTR of a transfer vector construct can be modified by substituting part or all of the transcriptional regulatory elements of the U3 region with heterologous enhancer/promoters. The changes were made to enhance the expression of transfer vector RNA in producer cells; to allow vector production in the absence of the HIV tat gene; and to remove the upstream wild-type copy of the HIV LTR that can recombine with the 3' deleted version to "rescue" the above described SIN vectors.

Thus, vectors containing the above-described alterations at the 5' LTR, 5' vectors, can find use as transfer vectors because of the sequences to enhance expression and in combination with packaging cells that do not express tat. Such 5' vectors can also carry modifications at the 3' LTR as discussed hereinabove to yield improved transfer vectors which have not only enhanced expression and can be used in packaging cells that do not express tat but can be self-inactivating as well.

The transcription from the HIV LTR is highly dependent on the transactivator function of the tat protein. In the presence of tat, often expressed by the core packaging construct existing in producer cells, vector transcription from the HIV LTR is stimulated strongly. As that full-length "viral" RNA has a full complement of packaging signals, the RNA is encapsidated efficiently into vector particles and transferred to target cells. The amount of vector RNA available for packaging in producer cells is a rate-limiting step in the production of infectious vector.

The entire enhancer or the entire enhancer and promoter regions of the 5' LTR can be substituted with the enhancer or the enhancer and promoter of the human cytomegalovirus (CMV) or murine Rous sarcoma virus (RSv).

The high level of expression of the 5' LTR modified transfer vector RNA obtained in producer cells in the absence of a packaging construct indicates the producing vector is functional in the absence of a functional tat gene. Functional deletion of the tat gene as indicated for the packaging plasmid disclosed hereinabove would confer a higher level of biosafety to the lentiviral vector system given the number of pathogenetic activities associated with the tat protein.

Exemplary Packaging Cell Lines

Pseudotyped lentiviral or retroviral particles can be produced by introducing a defective, recombinant lentiviral genome into a packaging cell (e.g., by infection with defective retroviral particle, or by other means for introducing DNA into a target cell, such as conventional transformation techniques). The defective retroviral genome minimally contains the long terminal repeats, the exogenous nucleotide sequence of interest to be transferred, and a packaging sequence (.phi.). In general, the packaging cell provides the missing retroviral components essential for retroviral replication, integration, and encapsidation, and also expresses a nucleotide sequence encoding the desired envelope protein. However, the packaging cell does not have all of the components essential for the production of retroviral particles. The nucleotide sequence(s) encoding the missing viral component(s) in the packaging cell can be either stably integrated into the packaging cell genome, and/or can be provided by a co-infecting helper virus.

The nucleotide sequences encoding the retroviral components and the lentiviral or retroviral RNA genome can be derived from any desired lenti- or retrovirus (e.g., murine, simian, avian, or human retroviruses). In general, the retroviral components can be derived from any retrovirus that can form pseudotyped retroviral particles with the desired envelope protein, e.g., VSV G. Where VSV G is the desired envelope protein, the retroviral components can be derived from MuLV, MoMLV, avian leukosis virus (ALV), human immunodeficiency virus (HIV), or any other retrovirus that can form pseudotyped virus with VSV G as the only envelope protein or with VSV G and a relatively small amount of retroviral envelope protein.

The present invention thus provides recombinant retroviral particles, particularly pseudotyped retroviral particles. Exemplary packaging cell lines are derived from 293, HeLa, Cf2Th, D17, MDCK, or BHK cells. Retroviral particles are preferentially produced by inducibly expressing an envelope protein of interest (e.g., a retroviral envelope or the envelope protein of vesicular stomatitis virus). Inducible expression of the envelope protein may be accomplished by operably linking an envelope protein-encoding nucleotide sequence to an inducible promoter (e.g., a promoter composed of a minimal promoter linked to at least one copy of tetO, the binding site for the tetracycline repressor (tetR) of the Escherichia coli tetracycline resistance operon Tn10). Expression from the inducible promoter is regulated by a transactivating factor, composed of a first ligand-binding domain that negatively regulates transcription from the inducible promoter (e.g., a prokaryotic tetracycline repressor polypeptide (tetR)). Transcription of the envelope-encoding nucleotide sequence under control of the inducible promoter is activated by a transactivator when tetracycline is absent.

The packaging cell line may comprise a first polynucleotide having an HIV genome operably linked to a first inducible promoter wherein the HIV genome is defective for cis-acting elements, for self-replication and for expression of functional Env protein; a second polynucleotide encoding a functional heterologous Env protein operably linked to a second inducible promoter; and a third polynucleotide encoding a regulatable transcriptional activator controlling transcription from the first and second inducible promoters.

In one embodiment, the first, second and third polynucleotides are contained in vectors. These polynucleotides can be contained in one or more vectors, preferably plasmid vectors. In an exemplary packaging cell line, the first polynucleotide is contained in a first plasmid vector and the second polynucleotide is contained in a second plasmid vector. The third polynucleotide encoding a regulatable transcriptional activator is exemplified herein as containing a minimal CMV immediate-early gene promoter linked to seven tandem copies of the tetR-binding site replacing the CMV promoter (BglII/BamHI fragment). As discussed herein, other viral envelopes and other indicator markers will be known to those of skill in the art for use in the present invention.

In one aspect of the invention, one or more polynucleotides encoding retroviral accessory proteins, are included as part of the first or second polynucleotide constructs, for example. Accessory proteins include vpr, vif, nef, vpx, tat, eve, and vpu.

Preferably, the transcriptional activator or transactivator can be expressed at high levels in a eukaryotic cell without significantly adversely affecting general cellular transcription in the host cell transactivator expression that is sufficient to facilitate transactivation of the inducible promoter, but that is not detrimental to the cell (e.g., is not toxic to the cell). "High Levels" can be a level of expression that allows detection of the transactivator by Western blot The transactivator can preferably be expressed in a wide variety of cell types, including mammalian and non-mammalian cells such as, but not limited to, human, monkey, mouse, hamster, cow, insect, fish, and frog cells.

The transactivator can be expressed either in vivo or in vitro, and expression of the transactivator can be controlled through selection of the promoter to which the nucleotide sequence encoding the transactivator is operably linked. For example, the promoter can be a constitutive promoter or an inducible promoter. Examples of such promoters include the human cytomegalovirus promoter IE (Boshart et al., 1985), ubiquitously expressing promoters such as HSV-Tk (McKnight et al., 1984) and .beta.-actin promoters (e.g., the human .beta.-actin promoter as described by Ng et al., 1985).

For example, where the transactivator is a tetR polypeptide, the inducible promoter is preferably a minimal promoter containing at least one tetO sequence, preferably at least 2 or more tandemly repeated tetO sequences, even more preferably at least 5 or more tandemly repeated tetO sequences, more preferably at least 7 tandemly repeated tetO sequences or more. The minimal promoter portion of the inducible promoter can be derived from any desired promoter, and is selected according to tet cell line in which the inducible expression system is to be used. Where the cell is a mammalian cell, a preferred minimal promoter is derived from CMV, preferably from the CMV immediate early gene 1A. In addition, other inducible promoters could be employed, such as the ecdysone-inducible promoters (Invitrogen Inc., San Diego, Calif.) or the lacZ inducible promoters.

The promoter of the transactivator can be a cell type-specific or tissue-specific promoter than preferentially facilitates transcription of the transactivator in a desired cell of tissue type. Exemplary cell type-specific and/or tissue specific promoters include promoters such as albumin (liver specific; Pinkert et al., 1987), lymphoid-specific promoters (Calame et al., 1988); in particular promoters of T-cell receptors (Winoto et al., 1989) and immunoglobulins (Banerji et al., 1983; Queen and Baltimore, 1983), neuron-specific promoters (e.g., the neurofilament promoter (Byrne et al., 1989), pancreas-specific promoters (Edlunch et al., 1985) or mammary gland-specific promoters (milk whey promoter, U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Promoters for expression of the transactivator can also be developmentally regulated promoters as the murine homeobox promoters (Kessel et al., 1990) or the .alpha.-fetoprotein promoter (Campes et al., 1989). The promoter can be used in combination with control regions allowing integration site independent expression of the transactivator (Grosveld et al., 1987). Preferably, the promoter is constitutive in the respective cell types. For instance, the promoter is a CMV promoter, preferably a CMV immediate early gene promoter.

Isolated Nucleic Acid-Based Vectors of the Invention

The isolated nucleic acid-based vectors of the invention, e.g., those which are not delivered in a viral particle and/or do not encode one or more viral proteins but may comprise viral transcriptional and/or translational regulatory elements, include a heterologous nucleic acid sequence of interest optionally operably linked to a regulatory nucleic acid sequence. The heterologous gene of interest in the isolated nucleic acid-based vector of the invention can be any nucleic acid of interest which can be transcribed. Generally the foreign gene encodes a polypeptide. Preferably the polypeptide has some therapeutic benefit. The polypeptide may supplement deficient or nonexistent expression of an endogenous protein in a host cell.

It may be desirable to modulate the expression of a gene regulating molecule in a cell by the introduction of a molecule by the method of the invention. The term "modulate" envisions the suppression of expression of a gene when it is over-expressed or augmentation of expression when it is under-expressed.

The method of the invention may also be useful for neuronal, glial, fibroblast or mesenchymal cell transplantation, or "grafting", which involves transplantation of transfected cells into the central nervous system or into the ventricular cavities or subdurally onto the surface of a host brain. Such methods for grafting will be known to those skilled in the art and are described in Neural Grafting in the Mammalian CNS, Bjorklund & Stenevi, eds. (1985).

For diseases due to deficiency of a protein product, gene transfer of an isolated nucleic acid-based vector of the invention could introduce a normal gene into the affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations.

The promoter sequence of an isolated nucleic acid-based vector of the invention may be homologous or heterologous to the desired gene sequence. A wide range of promoters may be utilized, including a viral or a mammalian promoter. Cell or tissue specific promoters can be utilized to target expression of gene sequences in specific cell populations. Suitable mammalian and viral promoters for the instant invention are available in the art.

Optionally during the cloning stage, the nucleic acid construct referred to as the transfer vector also contains a selectable marker gene. Marker genes are utilized to assay for the presence of the vector, and thus, to confirm infection and integration. The presence of a marker gene ensures the selection and growth of only those host cells which express the inserts. Typical selection genes encode proteins that confer resistance to antibiotics and other toxic substances, e.g., histidinol, puromycin, hygromycin, neomycin, methotrexate etc. and cell surface markers.

Exemplary Disorders and Genes

The invention includes the use of a vector, e.g., a lentiviral vector, comprising any open reading frame encoding a gene product useful to prevent, inhibit or treat a disorder in a mammal characterized by the lack of, or reduced levels of, that gene product. For example, the disorder may be characterized by the lack of, or reduced levels of one or more lysosomal enzymes (see, e.g., enzymes described in FIG. 5 in U.S. Pat. No. 5,798,366, the disclosure of which is specifically incorporated by reference herein). Exemplary disorders include GM1 gangliosidosis, which is caused by a deficiency in .beta.-galactosidase; Tay-Sachs disease, a GM2 gangliosidosis which is caused by a deficiency of .beta.-hexosaminidase A (acidic isozyme); Sandhoff disease, which is caused by a deficiency of .beta.-hexosaminidase A & B (acidic and basic isozymes); Fabry disease, which is caused by a deficiency in .alpha.-galactosidase; Hurler-syndrome, which is caused by a deficiency of alpha-L-iduronidase, mucopolysaccharidosis type VII, which is caused by a deficiency in beta-glucuronidase, and Gaucher disease, which is a deficiency in .beta.-glucocerebrosidase, as well as Hunter syndrome (a deficiency of iduronate-2-sulfatase); Sanfilippo syndrome (a deficiency of heparan sulfate sulfatase, N-acetylglucosamidase); Morquio syndrome (a deficiency of galactosamine-6-sulfate sulfatase or beta-galactosidase); Maroteaux-Lamy syndrome (a deficiency of arylsulfatase B); mucopolysaccharidosis type II; mucopolysaccharidosis type III (A, B, C or D; a deficiency of heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminide N-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IV (A or B; a deficiency of galactosamine-6-sulfatase and beta-galatacosidase); mucopolysaccharidosis type VI (a deficiency of arylsulfatase B); mucopolysaccharidosis type VII (a deficiency in beta-glucuronidase); mucopolysaccharidosis type VIII (a deficiency of glucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IX (a deficiency of hyaluronidase); Tay-Sachs disease (a deficiency in alpha subunit of beta-hexosaminidase); Sandhoff disease (a deficiency in both alpha and beta subunit of beta-hexosaminidase); GM1 gangliosidosis (type I or type II); Fabry disease (a deficiency in alpha galactosidase); metachromatic leukodystrophy (a deficiency of aryl sulfatase A); Pompe disease (a deficiency of acid maltase); fucosidosis (a deficiency of fucosidase); alpha-mannosidosis (a deficiency of alpha-mannosidase); beta-mannosidosis (a deficiency of beta-mannosidase), ceroid lipofuscinosis, and Gaucher disease (types I, II and III; a deficiency in glucocerebrosidase), as well as disorders such as Hermansky-Pudlak syndrome; Amaurotic idiocy; Tangier disease; aspartylglucosaminuria; congenital disorder of glycosylation, type Ia; Chediak-Higashi syndrome; macular dystrophy, corneal, 1; cystinosis, nephropathic; Fanconi-Bickel syndrome; Farber lipogranulomatosis; fibromatosis; geleophysic dysplasia; glycogen storage disease I; glycogen storage disease Ib; glycogen storage disease Ic; glycogen storage disease III; glycogen storage disease IV; glycogen storage disease V; glycogen storage disease VI; glycogen storage disease VII; glycogen storage disease 0; immunoosseous dysplasia, Schimke type; lipidosis; lipase b; mucolipidosis II; mucolipidosis II, including the variant form; mucolipidosis IV; neuraminidase deficiency with beta-galactosidase deficiency; mucolipidosis I; Niemann-Pick disease (a deficiency of sphingomyelinase); Niemann-Pick disease without sphingomyelinase deficiency (a deficiency of npc1, a cholesterol metabolizing enzyme); Refsum disease; Sea-blue histiocyte disease; infantile sialic acid storage disorder; sialuria; multiple sulfatase deficiency; triglyceride storage disease with impaired long-chain fatty acid oxidation; Winchester disease; Wolman disease (a deficiency of cholesterol hydrolase); Deoxyribonuclease I-like 1 disorder; arylsulfatase E disorder; ATPase, H+ transporting, lysosomal, subunit 1 disorder; glycogen storage disease IIb; Ras-associated protein rab9 disorder; chondrodysplasia punctata 1, X-linked recessive disorder; glycogen storage disease VIII; lysosome-associated membrane protein 2 disorder; Menkes syndrome; congenital disorder of glycosylation, type Ic; and sialuria. In particular, the invention is useful to prevent, inhibit or treat lysosomal storage diseases wherein the lysosomal enzyme is trafficked to the lysosome (within the cell and between cells) by specific glycosylation.

For instance, Tay-Sachs disease results from mutations in the HexA gene, which encodes the alpha subunit of .beta.-hexosaminidase, leading to a deficiency in the A isoenzyme. The A isoenzyme is responsible for the degradation of GM2 ganglioside. When this enzyme is deficient in humans, GM2 ganglioside accumulates progressively and leads to severe neurological degeneration. In the mouse model of Tay-Sachs disease (generated by the targeted disruption of the HexA gene) (Sandhoff et al., 1989), the mice store GM2 ganglioside in a progressive fashion, but the levels never exceed the threshold required to elicit neurodegeneration. In the mouse (but not in a human) a sialidase is sufficiently abundant that it can convert GM2 to GA2 (asialo ganglioside 2), which can then be catabolized by the hexosaminidase B isoenzyme.

Gaucher disease is the name given to a group of lysosomal storage disorders caused by mutations in the gene that codes for an enzyme called glucocerebrosidase ("GC"). Gaucher disease is caused by deficiency of GC. All of the mutations in the gene alter the structure and function of the enzyme which lead to an accumulation of the undegraded glycolipid substrate glucosylceramide, also called glucocerebroside, in cells of the reticuloendothelial system. Each particular mutation of the human GC gene leads to a clinical disease collectively known as Gaucher disease. These disorders are usually classified into three types; type 1 (non-neuronopathic), type 2 (acute neuronopathic) and type 3 (subacute neuronopathic), the type depending on the presence and severity of neurologic involvement.

GC is a monomeric, membrane-associated, hydrophobic glycoprotein with a molecular weight of 65,000 daltons. Human GC contains 497 amino acids and is translated as a precursor protein with a 19 amino acid hydrophobic signal peptide which directs its co-translational insertion into the lumen of the endoplasmic reticulum-golgi-lysosome complex as reported by Erickson et al. (1985). GC acts at the acidic pH of the lysosome to hydrolyze beta-glucosidic linkages in complex lipids ubiquitously found in all membranes to form the byproducts of glucose and ceramide. The catalytic activity of GC is increased in vitro by detergents, lipids, and in vivo by a naturally occurring activator known as sphingolipid activator protein-2 (SAP-2 or saposin C). See, Ho et al. (1971); O'Brian et al. (1988). While more than twenty mutations in the human GC gene are known, only two are common. See, Tsuji et al. (1988). The two common mutations account for approximately 70% of the mutant alleles, as reported by Firon et al. (1990). Mutant GC genes code for aberrant proteins that are either catalytically altered or unstable and rapidly disappear from the cell.

Although GC is deficient in all of a subject's cells, for unknown reasons, the accumulation of the substrate glucosylceramide occurs virtually only in macrophages. To correct the enzyme deficiency in macrophages, two approaches have been used. The first treatment is based allogeneic bone marrow transplantation, which results in the repopulation of affected tissues with enzyme-competent macrophages. See, Rappeport et al. (1986). The second approach to treatment which has resulted in clinical improvement in Gaucher disease patients is macrophage-targeted enzyme replacement. This treatment takes advantage of naturally occurring mannose receptors on macrophages and the exposition of accessible mannose receptors in the oligosaccharides of glucocerebrosidase to efficiently deliver the enzyme to macrophages. See, Barranger (1989); Takasaki et al. (1984); and Furbish et al., (1981). However, allogeneic bone marrow transplantation has associated with it morbidity and mortality risks that are unacceptable for many patients. Further, HLA matched bone marrow donors do not exist for the majority of patients. As for macrophage-targeted enzyme replacement, it is currently an expensive and life-long therapy.

Hurler syndrome is an autosomal recessive disease resulting from deficient alpha-iduronidase enzymatic activity and the consequent systemic accumulation of glycosaminoglycan (GAG) substrates. The disease is characterized by hepatosplenomegaly, severe skeletal involvement, progressive mental retardation, and is typically lethal in childhood.

To be an effective permanent treatment for any disease capable of being treated by gene therapy, the transfer and sustained expression of genes in cells important to the pathogenesis of the particular disease is required. Sufficient and long term expression of a transduced gene in the progeny of transduced cells, e.g., transduced stem cells such as pluripotent bone marrow stem cells, for example, using a lentivirus, could correct the deficiency of the enzyme in many if not all relevant cell types.

Dosages, Formulations and Routes of Administration of the Agents of the Invention

The therapeutic agents of the invention are preferably administered so as to achieve beneficial results. The amount administered will vary depending on various factors including, but not limited to, the agent chosen, the disease, whether prevention or treatment is to be achieved, and if the agent is modified for bioavailability and in vivo stability.

Administration of sense or antisense nucleic acid molecule may be accomplished through the introduction of cells transformed with an expression cassette comprising the nucleic acid molecule (see, for example, WO 93/02556) or the administration of the nucleic acid molecule (see, for example, Felgner et al., U.S. Pat. No. 5,580,859, Pardoll et al., Immunity, 3, 165 (1995); Stevenson et al., Immunol. Rev., 145, 211 (1995); Molling, J. Mol. Med., 75, 242 (1997); Donnelly et al., Ann. N.Y. Acad. Sci., 772, 40 (1995); Yang et al., Mol. Med. Today, 2, 476 (1996); Abdallah et al., Biol. Cell, 85, 1 (1995)). Pharmaceutical formulations, dosages and routes of administration for nucleic acids are generally disclosed, for example, in Felgner et al., supra. Nucleic acid molecules may be complexed with polyethyleneimine, polylysine or cationic lipids such as DOTMA, DOTAP, DOGS, or DC-Chol (N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium chloride, DOTAP; N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride, DOTMA; 3.beta.-[N--(N',N'-Dimethylaminoethane)-carbamoyl]Cholesterol, DC-Chol) In one embodiment, DNA is delivered under pressure into the hepatic circulation.

The amount of therapeutic agent administered is selected to treat a particular indication. The therapeutic agents of the invention are also amenable to chronic use for prophylactic purposes, preferably by systemic administration.

Administration of the therapeutic agents in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.

One or more suitable unit dosage forms comprising the therapeutic agents of the invention, which, as discussed below, may optionally be formulated for sustained release, can be administered by a variety of routes including oral, or parenteral, including by rectal, buccal, vaginal and sublingual, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intrapulmonary and intranasal routes. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

When the therapeutic agents of the invention are prepared for oral administration, they are preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation. By "pharmaceutically acceptable" it is meant the carrier, diluent, excipient, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient hereof The active ingredient for oral administration may be present as a powder or as granules; as a solution, a suspension or an emulsion; or in achievable base such as a synthetic resin for ingestion of the active ingredients from a chewing gum. The active ingredient may also be presented as a bolus, electuary or paste.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, douches, lubricants, foams or sprays containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. Formulations suitable for rectal administration may be presented as suppositories.

Pharmaceutical formulations containing the therapeutic agents of the invention can be prepared by procedures known in the art using well known and readily available ingredients. For example, the agent can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include the following fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose, HPMC and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.

For example, tablets or caplets containing the agents of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Caplets and tablets can also include inactive ingredients such as cellulose, pregelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, and zinc stearate, and the like. Hard or soft gelatin capsules containing an agent of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric coated caplets or tablets of an agent of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.

The therapeutic agents of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.

The pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

These formulations can contain pharmaceutically acceptable vehicles and adjuvants which are well known in the prior art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol", polyglycols and polyethylene glycols, C.sub.1-C.sub.4 alkyl esters of short-chain acids, preferably ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol", isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.

The compositions according to the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They can also contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like.

It is possible to add, if necessary, an adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes and colorings. Also, other active ingredients may be added, whether for the conditions described or some other condition.

For example, among antioxidants, t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and .alpha.-tocopherol and its derivatives may be mentioned. The galenical forms chiefly conditioned for topical application take the form of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, or alternatively the form of aerosol formulations in spray or foam form or alternatively in the form of a cake of soap.

Additionally, the agents are well suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal or respiratory tract, possibly over a period of time. The coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, and the like.

The therapeutic agents of the invention can be delivered via patches for transdermal administration. See U.S. Pat. No. 5,560,922 for examples of patches suitable for transdermal delivery of a therapeutic agent. Patches for transdermal delivery can comprise a backing layer and a polymer matrix which has dispersed or dissolved therein a therapeutic agent, along with one or more skin permeation enhancers. The backing layer can be made of any suitable material which is impermeable to the therapeutic agent. The backing layer serves as a protective cover for the matrix layer and provides also a support function. The backing can be formed so that it is essentially the same size layer as the polymer matrix or it can be of larger dimension so that it can extend beyond the side of the polymer matrix or overlay the side or sides of the polymer matrix and then can extend outwardly in a manner that the surface of the extension of the backing layer can be the base for an adhesive means Alternatively, the polymer matrix can contain, or be formulated of, an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long-term applications it might be desirable to use microporous and/or breathable backing laminates, so hydration or maceration of the skin can be minimized.

Examples of materials suitable for making the backing layer are films of high and low density polyethylene, polypropylene, polyurethane, polyvinylchloride, polyesters such as poly(ethylene phthalate), metal foils, metal foil laminates of such suitable polymer films, and the like. Preferably, the materials used for the backing layer are laminates of such polymer films with a metal foil such as aluminum foil. In such laminates, a polymer film of the laminate will usually be in contact with the adhesive polymer matrix.

The backing layer can be any appropriate thickness which will provide the desired protective and support functions. A suitable thickness will be from about 10 to about 200 microns.

Generally, those polymers used to form the biologically acceptable adhesive polymer layer are those capable of forming shaped bodies, thin walls or coatings through which therapeutic agents can pass at a controlled rate. Suitable polymers are biologically and pharmaceutically compatible, nonallergenic and insoluble in and compatible with body fluids or tissues with which the device is contacted. The use of soluble polymers is to be avoided since dissolution or erosion of the matrix by skin moisture would affect the release rate of the therapeutic agents as well as the capability of the dosage unit to remain in place for convenience of removal.

Exemplary materials for fabricating the adhesive polymer layer include polyethylene, polypropylene, polyurethane, ethylene/propylene copolymers, ethylene/ethylacrylate copolymers, ethylene/vinyl acetate copolymers, silicone elastomers, especially the medical-grade polydimethylsiloxanes, neoprene rubber, polyisobutylene, polyacrylates, chlorinated polyethylene, polyvinyl chloride, vinyl chloride-vinyl acetate copolymer, crosslinked polymethacrylate polymers (hydrogel), polyvinylidene chloride, poly(ethylene terephthalate), butyl rubber, epichlorohydrin rubbers, ethylenevinyl alcohol copolymers, ethylene-vinyloxyethanol copolymers; silicone copolymers, for example, polysiloxane-polycarbonate copolymers, polysiloxane-polyethylene oxide copolymers, polysiloxane-polymethacrylate copolymers, polysiloxane-alkylene copolymers (e.g., polysiloxane-ethylene copolymers), polysiloxane-alkylenesilane copolymers (e.g., polysiloxane-ethylenesilane-copolymers), and the like; cellulose polymers, for example methyl or ethyl cellulose, hydroxy propyl methyl cellulose, and cellulose esters; polycarbonates; polytetrafluoroethylene; and the like.

Preferably, a biologically acceptable adhesive polymer matrix should be selected from polymers with glass transition temperatures below room temperature. The polymer may, but need not necessarily, have a degree of crystallinity at room temperature. Cross-linking monomeric units or sites can be incorporated into such polymers. For example, cross-linking monomers can be incorporated into polyacrylate polymers, which provide sites for cross-linking the matrix after dispersing the therapeutic agent into the polymer. Known cross-linking monomers for polyacrylate polymers include polymethacrylic esters of polyols such as butylene diacrylate and dimethacrylate, trimethylol propane trimethacrylate and the like. Other monomers which provide such sites include allyl acrylate, allyl methacrylate, diallyl maleate and the like.

Preferably, a plasticizer and/or humectant is dispersed within the adhesive polymer matrix. Water-soluble polyols are generally suitable for this purpose. Incorporation of a humectant in the formulation allows the dosage unit to absorb moisture on the surface of skin which in turn helps to reduce skin irritation and to prevent the adhesive polymer layer of the delivery system from failing.

Therapeutic agents released from a transdermal delivery system must be capable of penetrating each layer of skin. In order to increase the rate of permeation of a therapeutic agent, a transdermal drug delivery system must be able in particular to increase the permeability of the outermost layer of skin, the stratum corneum, which provides the most resistance to the penetration of molecules. The fabrication of patches for transdermal delivery of therapeutic agents is well known to the art.

For administration to the upper (nasal) or lower respiratory tract by inhalation, the therapeutic agents of the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatine or blister packs from which the powder may be administered with the aid of an inhalator, insufflator or a metered-dose inhaler.

For intra-nasal administration, the therapeutic agent may be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).

The local delivery of the therapeutic agents of the invention can also be by a variety of techniques which administer the agent at or near the site of disease. Examples of site-specific or targeted local delivery techniques are not intended to be limiting but to be illustrative of the techniques available. Examples include local delivery catheters, such as an infusion or indwelling catheter, e.g., a needle infusion catheter, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct applications.

For topical administration, the therapeutic agents may be formulated as is known in the art for direct application to a target area. Conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols, as well as in toothpaste and mouthwash, or by other suitable forms, e.g., via a coated condom. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The active ingredients can also be delivered via iontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122; 4,383,529; or 4,051,842. The percent by weight of a therapeutic agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01% to 95% of the total weight of the formulation, and typically 0.1-25% by weight.

When desired, the above-described formulations can be adapted to give sustained release of the active ingredient employed, e.g., by combination with certain hydrophilic polymer matrices, e.g., comprising natural gels, synthetic polymer gels or mixtures thereof.

Drops, such as eye drops or nose drops, may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.

The therapeutic agent may further be formulated for topical administration in the mouth or throat. For example, the active ingredients may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; mouthwashes comprising the composition of the present invention in a suitable liquid carrier; and pastes and gels, e.g., toothpastes or gels, comprising the composition of the invention.

The formulations and compositions described herein may also contain other ingredients such as antimicrobial agents, or preservatives. Furthermore, the active ingredients may also be used in combination with other therapeutic agents.
 

Claim 1 of 10 Claims

1. A method to prevent or inhibit a disorder characterized by the absence or reduced levels of a alpha-L-iduronidase in a mammal, comprising: administering to a vascular compartment of a mammal having or at risk of the disorder, an effective amount of a recombinant lentivirus comprising a nucleic acid segment encoding alpha-L-iduronidase

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