US20040022766A1 - Method of treating or retarding the development of blindness - Google Patents

Method of treating or retarding the development of blindness Download PDF

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Publication number
US20040022766A1
US20040022766A1 US10/300,720 US30072002A US2004022766A1 US 20040022766 A1 US20040022766 A1 US 20040022766A1 US 30072002 A US30072002 A US 30072002A US 2004022766 A1 US2004022766 A1 US 2004022766A1
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Prior art keywords
gene
cells
normal
ocular
promoter
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Gregory Acland
Gustayo Aguirre
Jean Bennett
William Hauswirth
Samuel Jacobson
Albert Maguire
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FLORIDA RESEARCH FOUNDATION INCORPORATED UNIVERSITY OF
Cornell Research Foundation Inc
University of Pennsylvania Penn
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Assigned to FLORIDA RESEARCH FOUNDATION INCORPORATED, UNIVERSITY OF reassignment FLORIDA RESEARCH FOUNDATION INCORPORATED, UNIVERSITY OF ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FLORIDA, UNIVERSITY OF
Assigned to CORNELL RESEARCH FOUNDATION INC. reassignment CORNELL RESEARCH FOUNDATION INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AGUIRRE, GUSTAVO D., ACLAND, GREGORY M.
Assigned to UNIVERSITY OF FLORIDA reassignment UNIVERSITY OF FLORIDA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HAUSWIRTH, WILLIAM W.
Publication of US20040022766A1 publication Critical patent/US20040022766A1/en
Priority to US11/511,201 priority patent/US20070077228A1/en
Assigned to THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA reassignment THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BENNETT, JEAN, MAGUIRE, ALBERT M.
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF PENNSYLVANIA
Priority to US12/253,955 priority patent/US20090074723A1/en
Priority to US12/832,282 priority patent/US8147823B2/en
Priority to US13/406,666 priority patent/US20120225930A1/en
Priority to US13/766,267 priority patent/US20130216500A1/en
Priority to US14/310,015 priority patent/US9433688B2/en
Priority to US15/146,274 priority patent/US20160263246A1/en
Priority to US15/220,937 priority patent/US20170014529A1/en
Abandoned legal-status Critical Current

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Definitions

  • the invention relates generally to the use of recombinant viruses to deliver a desired transgene to retinal pigment epithelial cells of patients suffering from retinal degenerative diseases.
  • RPE retinal pigment epithelium
  • U.S. Pat. No. 5,827,702 refers to methods for generating a genetically engineered ocular cell by contacting the cell with an exogenous nucleic acid under conditions in which the exogenous nucleic acid is taken up by the cell for expression.
  • the exogenous nucleic acid is described as a retrovirus, an adenovirus, an adeno-associated virus or a plasmid. See, also, International Patent Publications WO 00/15822, published Mar. 23, 2000 and WO 98/48097, published Oct. 29, 1998.
  • the invention provides a method for treating an ocular disorder in a human or animal subject characterized by the defect or absence of a normal gene in the ocular cells.
  • the method includes administering to the subject by subretinal injection an effective amount of a recombinant adeno-associated virus carrying a nucleic acid sequence encoding the normal gene under the control of a promoter sequence which expresses the product of the gene in the ocular cells.
  • the invention provides a method for treating an ocular disorder in a human or animal subject characterized by the defect or absence of a normal gene in the retinal pigment epithelial (RPE) cells of the subject.
  • the method involves administering to the subject by subretinal injection an effective amount of a recombinant virus carrying a nucleic acid sequence encoding a normal retinal pigment epithelial (RPE) cell-specific gene under the control of a promoter sequence which expresses the product of the gene in RPE cells.
  • the gene is the RPE65 gene.
  • the invention provides a method for treating Leber congenital amaurosis in a subject by administering to the subject by subretinal injection an effective amount of a recombinant virus carrying a nucleic acid sequence encoding a normal gene under the control of a promoter sequence which expresses the product of the gene in ocular cells, wherein the cells contain a mutated version of the gene. Expression of the normal gene provides to the cells the product necessary to restore or maintain vision in the subject.
  • the cells are RPE or photoreceptor cells, and the promoters are cell-specific promoters.
  • the invention provides a composition for treatment of an ocular disorder characterized by the defect or absence of a normal gene in the ocular cells of the subject.
  • Such compositions comprise effective amounts of a recombinant adeno-associated virus carrying a nucleic acid sequence encoding the normal gene under the control of a promoter sequence which expresses the product of the gene in the ocular cells, formulated with a carrier and additional components suitable for subretinal injection.
  • the normal gene is RPE65.
  • the invention provides a method for treating an ocular disorder in a human, other mammalian or other animal subject.
  • the ocular disorder is one which involves a mutated or absent gene in a retinal pigment epithelial cell or a photoreceptor cell.
  • the method of this invention comprises the step of administering to the subject by subretinal injection an effective amount of a recombinant virus carrying a nucleic acid sequence encoding an ocular cell-specific normal gene operably linked to, or under the control of, a promoter sequence which directs the expression of the product of the gene in the ocular cells and replaces the lack of expression or incorrect expression of the mutated or absent gene.
  • this method is useful for the treatment and/or restoration of at least partial vision to subjects that have lost vision due to ocular disorders, such as RPE-associated retinopathies, which are characterized by a long-term preservation of ocular tissue structure despite loss of function and by the association between function loss and the defect or absence of a normal gene in the ocular cells of the subject.
  • ocular disorders such as RPE-associated retinopathies
  • a variety of such ocular disorders are known, such as childhood onset blinding diseases, retinitis pigmentosa, macular degeneration, and diabetic retinopathy, as well as ocular blinding diseases known in the art.
  • the particular ocular disorder treated by this method may include the above-mentioned disorders and a number of diseases which have yet to be so characterized.
  • the particular ocular disorder being treated in the examples is Leber congenital amaurosis, which affects humans.
  • this invention is not limited to the treatment of that disorder alone.
  • LCA Leber congenital amaurosis
  • RPE retinal pigment epithelium
  • RPE65 A naturally-occurring large animal model of an analogous severe disease of retinal degenerations is the RPE65 mutant dog.
  • LCA causes near total blindness from early in life.
  • RPE65 is an evolutionarily-conserved 65 kDa membrane-associated protein (Redmond, T. & Hamel, C. 2000 Meth. Enzymol . 317, 705-724 and Bavik, C. et al, 1992 J. Biol.
  • RPE65 deficiency in mice results in accumulation of all-trans-retinyl esters, undetectable levels of rhodopsin, rod photoreceptor dysfunction, inclusions in the RPE, and slow retinal degeneration.
  • the compound 9-cis-retinal can restore visual pigment and function in RPE65-deficient mice (Redmond, T., et al. 1998 Nat. Genet 20, 344-351 and Van Hooser, J. P., et al. 2000 Proc. Natl Acad Sci USA 97, 8623-8628).
  • the RPE65 mutant dog shows early and severe visual impairment caused by a homozygous 4 bp-deletion in the RPE65 gene.
  • the deletion results in a frame shift leading to a premature stop codon, eliminating more than two-thirds of the wildtype polypeptide.
  • Histopathology in homozygotes shows prominent RPE inclusions and slightly abnormal rod photoreceptor morphology present within the first year of life, and slowly progressive photoreceptor degeneration in older dogs. See, e.g., Wrigstad, A. Hereditary Dystrophy of the Retina and the Retinal Pigment Epithelium in a Strain of Briard Dogs: A Clinical, Morphological and Electrophysiological Study. Linkoping University Medical Dissertations (1994); Narfstrom, K. et al, 1989 Brit J Ophthalmol . 73, 750-756; and Aguirre, G., et al. 1998 Mol. Vis. 4, 23.
  • recombinant viruses such as recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant retroviruses, recombinant poxviruses, and other known viruses in the art, as well as plasmids, cosmids and phages, etc.
  • AAV recombinant adeno-associated virus
  • retroviruses recombinant retroviruses
  • poxviruses recombinant poxviruses
  • other known viruses in the art as well as plasmids, cosmids and phages, etc.
  • the vector is a recombinant AAV carrying a wildtype (i.e., normal) version of a selected transgene-encoding cDNA driven by a promoter that expresses the product of the wildtype cDNA in selected ocular cells of the affected subject.
  • Adeno-associated viruses are small, single-stranded DNA viruses which require helper virus to facilitate efficient replication (K. I. Berns, Parvoviridae: the viruses and their replication , p. 1007-1041, in F. N. Fields et al., Fundamental Virology, 3rd ed., vol. 2, (Lippencott-Raven Publishers, Philadelphia, Pa.) (1995)).
  • the 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITR) and two open reading frames which encode the Rep proteins and Cap proteins, respectively.
  • the Rep reading frame encodes four proteins of molecular weight 78 kD, 68 kD, 52 kD and 40 kD.
  • the Cap reading frame encodes three structural proteins of molecular weight 85 kD (VP 1), 72 kD (VP2) and 61 kD (VP3) (Berns, cited above) which form the virion capsid. More than 80% of total proteins in AAV virion comprise VP3.
  • Flanking the rep and cap open reading frames at the 5′ and 3′ ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures.
  • ITRs inverted terminal repeats
  • the two ITRs are the only cis elements essential for AAV replication, rescue, packaging and integration of the AAV genome.
  • AAVs have been found in many animal species, including primates, canine, fowl and human (F. A. Murphy et al., “The Classification and Nomenclature of Viruses: Sixth Report of the International Committee on Taxonomy of Viruses”, Archives of Virology, (Springer-Verlag, Vienna) (1995)).
  • Six primate serotypes have been reported (AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6).
  • the AAV ITR sequences and other AAV sequences employed in generating the minigenes, vectors, and capsids, and other constructs used in the present invention may be obtained from a variety of sources.
  • sequences may be provided by AAV type 5, AAV type 2, AAV type 1, AAV type 3, AAV type 4, AAV type 6, or other AAV serotypes or other densoviruses, including presently identified human AAV types and AAV serotypes yet to be identified.
  • AAVs known to infect other animals may also provide these ITRs employed in the molecules or constructs of this invention.
  • the capsids from a variety of serotypes of AAV may be “mixed and matched” with the other vector components. See, e.g., International Patent Publication No. WO01/83692, published Nov. 8, 2001, and incorporated herein by reference.
  • a variety of these viral serotypes and strains are available from the American Type Culture Collection, Manassas, Va., or are available from a variety of academic or commercial sources.
  • the source of the sequences utilized in preparation of the constructs of the invention is not a limitation of the present invention.
  • the selection of the species and serotype of AAV that provides these sequences is within the skill of the artisan and does not limit the following invention.
  • the AAV sequences are typically in the form of a rAAV construct (e.g., a minigene or cassette) which is packaged into a rAAV virion.
  • a rAAV construct e.g., a minigene or cassette
  • the rAAV minigene useful in this invention is formed by AAV inverted terminal repeat sequences (ITRs) and a heterologous molecule for delivery to a host cell.
  • the minigene contains AAV 5′ ITRs and 3′ ITRs located 5′ and 3′ to the heterologous molecule, respectively.
  • the minigene may be desirable for the minigene to contain the 5′ ITR and 3′ ITR sequences arranged in tandem, e.g., 5′ to 3′ or a head-to-tail, or in another alternative configuration.
  • the ITRs sequences may be located immediately upstream and/or downstream of the heterologous molecule, or there may be intervening sequences.
  • the ITRs may be selected from AAV5, or from among the other AAV serotypes, as described herein.
  • a minigene may contain 5′ ITRs from one serotype and 3′ ITRs from a second serotype.
  • the AAV sequences employed are preferably the 145 bp cis-acting 5′ and 3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, cited above).
  • the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning.
  • the heterologous molecule may be any substance which is desired to be delivered to a cell, including, without limitation, a polypeptide, protein, enzyme, carbohydrate, chemical moiety, or nucleic acid sequences which may include oligonucleotides, RNA, and/or DNA.
  • the heterologous molecule is a selected transgene under the control of a selected promoter and other conventional vector regulatory components. See, e.g., U.S. Pat. Nos. 5,856,152 and 5,871,982.
  • the heterologous molecule may be a nucleic acid molecule which introduces specific genetic modifications into human chromosomes, e.g., for correction of mutated genes. See, e.g., D. W. Russell & R. K. Hirata, 1998 Nat. Genet ., 18:325-330.
  • the heterologous molecule is a nucleic acid molecule is a transgene.
  • transgene refers to a nucleic acid sequence heterologous to the AAV sequence, encoding a desired product, e.g., a polypeptide or protein of interest, and the regulatory sequences which direct transcription and/or translation thereof in a host cell, and permit expression of the encoded product in a host cell. Suitable encoded products and regulatory sequences are discussed in more detail below.
  • the selection of the heterologous molecule delivered by the AAV minigene is not a limitation of the present invention.
  • the ocular cells which are the target of the treatment method are the retinal pigment epithelial (RPE) cells.
  • RPE retinal pigment epithelial
  • the specific gene which is mutated or absent in the disorder may be the RPE65 gene.
  • Another gene which is mutated or absent in the disorder in humans may be the arylhydrocarbon-interacting receptor protein like 1 (AIPL1).
  • the normal gene i.e., the transgene
  • the normal gene is the normal, species-matched version of the mutated gene, e.g., wildtype canine RPE65 for the treatment of canine LCA or wildtype human RPE65 for the treatment of human LCA, wildtype human AIPL1 for the treatment of a certain type of human blinding diseases, etc.
  • the gene can be the CRB1 (RP12) gene.
  • the transgene can be the lecithin retinal acetyltransferase (LRAT) gene.
  • LRAT lecithin retinal acetyltransferase
  • the ocular cells which are the target of the treatment method are the photoreceptor cells.
  • the specific gene which is mutated or absent in the disorder may be the photoreceptor-specific homeo box gene (CRX).
  • the specific gene which is mutated or absent in the disorder may be the retinal guanylate cyclase gene (GUCY2D).
  • the transgene is a nucleotide sequence encoding RPGR Interacting Protein 1 (RPGRIP1).
  • the normal gene, i.e., the transgene, present in the recombinant adeno-associated virus is the normal, species-matched version of the mutated gene, e.g., wildtype murine CRX for the treatment of the correlative murine blinding disorder or wildtype human CRX for the treatment of the correlative human blinding disorder, wildtype chicken GUCY2D for the treatment of the correlative chicken blinding disorder or wildtype human GUCY2D for the treatment of the correlative human blinding disorder, etc.
  • These transgenes may be obtained from conventional sources, e.g., from university laboratories or depositories, or synthesized from information obtained from Genbank by known techniques.
  • LCA3 located at chromosome 14q24 and LCA5, located at chromosome 6q11-q16, among others.
  • transgene Genes responsible for disorders other than LCA may also be employed as the transgene, as suitable ocular diseases are identified. Thus, different transgene may be used in the design of similar vectors of this invention for the treatment of disorders other than LCA.
  • genes which may be absent or mutated in the blinding disorders identified above include dystrophin, ABCR, EMP1, TIMP3, MERTCK and ELOVL4.
  • One or more of the wildtypes of these genes may be administered to ocular cells, particularly the RPE, in the same manner as is the exemplified RPE65 for the treatment of LCA.
  • One of skill in the art may obtain the appropriate gene sequences and design the appropriate vectors for such use in view of this disclosure and in view of other information known to the art.
  • a different transgene may be used to encode each subunit of a protein, or to encode different peptides or proteins. This is desirable when the size of the DNA encoding the protein subunit is large, e.g., for an immunoglobulin, the platelet-derived growth factor, or a dystrophin protein.
  • a cell is infected with the recombinant virus containing each of the different subunits.
  • different subunits of a protein may be encoded by the same transgene.
  • a single transgene includes the DNA encoding each of the subunits, with the DNA for each subunit separated by an internal ribozyme entry site (IRES).
  • IRES internal ribozyme entry site
  • other methods which do not require the use of an IRES may be used for co-expression of proteins. Such other methods may involve the use of a second internal promoter, an alternative splice signal, a co- or post-translational proteolytic cleavage strategy, among others which are known to those of skill in the art.
  • the minigene or transgene includes appropriate sequences that are operably linked to the nucleic acid sequences encoding the product of interest to promote its expression in a host cell.
  • “Operably linked” sequences present in the minigene include both expression control sequences (e.g. promoters) that are contiguous with the coding sequences for the product of interest and expression control sequences that act in trans or at a distance to control the expression of the product of interest.
  • expression control sequences e.g. promoters
  • expression control sequences e.g. promoters
  • the regulatory elements described herein may also be used in other heterologous molecules and the other constructs described in this application.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein processing and/or secretion.
  • efficient RNA processing signals such as splicing and polyadenylation signals
  • sequences that stabilize cytoplasmic mRNA sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein processing and/or secretion.
  • a great number of expression control sequences e.g., native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized to drive expression of the gene, depending upon the type of expression desired.
  • expression control sequences typically include a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, cytomegalovirus, etc., and a polyadenylation sequence which may include splice donor and acceptor sites.
  • the polyadenylation sequence generally is inserted following the transgene sequences and before the 3′ ITR sequence.
  • the bovine growth hormone polyA used.
  • the regulatory sequences useful in the constructs of the present invention may also contain an intron, desirably located between the promoter/enhancer sequence and the gene.
  • an intron sequence is also derived from SV-40, and is referred to as the SV-40 T intron sequence.
  • Another suitable sequence includes the woodchuck hepatitis virus post-transcriptional element. (See, e.g., L. Wang and I. Verma, 1999 Proc. Natl. Acad. Sci., USA , 96:3906-3910).
  • IRES internal ribosome entry site
  • An IRES sequence or other suitable systems as are discussed above, may be used to produce more than one polypeptide from a single gene transcript.
  • An IRES (or other suitable sequence) is used to produce a protein that contains more than one polypeptide chain or to express two different proteins from or within the same cell.
  • An exemplary IRES is the poliovirus internal ribosome entry sequence, which supports transgene expression in photoreceptors, RPE and ganglion cells.
  • the IRES is located 3′ to the transgene in the rAAV vector.
  • the selection of the promoter to be employed in the rAAV may be made from among a wide number of constitutive or inducible promoters that can express the selected transgene in an ocular.
  • the promoter is cell-specific.
  • the term “cell-specific” means that the particular promoter selected for the recombinant vector can direct expression of the selected transgene is a particular ocular cell type.
  • the promoter is specific for expression of the transgene in RPE cells.
  • the promoter is specific for expression of the transgene in photoreceptor cells.
  • Examples of constitutive promoters which may be included in the rAAV of this invention include, without limitation, the exemplified CMV immediate early enhancer/chicken ⁇ -actin (C ⁇ A) promoter-exon 1-intron 1 element of Example 1, the RSV LTR promoter/enhancer, the SV40 promoter, the CMV promoter, the dihydrofolate reductase promoter, and the phosphoglycerol kinase (PGK) promoter.
  • C ⁇ A CMV immediate early enhancer/chicken ⁇ -actin
  • PGK phosphoglycerol kinase
  • RPE-specific promoters include, for example, the RPE-65 promoter, the tissue inhibitor of metalloproteinase 3 (Timp3) promoter, and the tyrosinase promoter. Still other RPE-specific promoters are known to those of skill in the art. See, e.g., the promoters described in International Patent Publication WO 00/15822.
  • photoreceptor specific promoters include, without limitation, the rod opsin promoter, the red-green opsin promoter, the blue opsin promoter, the inter photoreceptor binding protein (IRBP) promoter and the cGMP- ⁇ -phosphodiesterase promoter. See, e.g., the promoters described in International Patent Publication WO 98/48097.
  • an inducible promoter is employed to express the transgene product, so as to control the amount and timing of the ocular cell's production thereof.
  • Such promoters can be useful if the gene product proves to be toxic to the cell upon excessive accumulation.
  • Inducible promoters include those known in the art and those discussed above including, without limitation, the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7 promoter; the ecdysone insect promoter; the tetracycline-repressible system; the tetracycline-inducible system; the RU486-inducible system; and the rapamycin-inducible system.
  • Any type of inducible promoter which is tightly regulated and is specific for the particular target ocular cell type may be used.
  • Other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particularly differentiation state of the cell, or in replicating cells only.
  • Such selection is a routine matter and is not a limitation of the molecule or construct. For instance, one may select one or more expression control sequences, operably link the sequence to a transgene of interest, and insert the “minigene” comprising the expression control sequence and the transgene into an AAV vector.
  • the vector may be packaged into an infectious particle or virion following one of the methods for packaging the rAAV taught in the art.
  • the rAAV virus of the invention may be constructed and produced using the materials and methods described herein, as well as those known to those of skill in the art.
  • Such engineering methods used to construct any embodiment of this invention are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, and Ausubel et al., cited above; and International Patent Application No. W095/13598.
  • methods suitable for producing a rAAV cassette in an adenoviral capsid have been described in U.S. Pat. Nos. 5,856,152 and 5,871,982.
  • a host cell in order to package the rAAV construct into a rAAV virion, a host cell must contain sequences necessary to express AAV rep and AAV cap or functional fragments thereof as well as helper genes essential for AAV production.
  • the rep78/52 proteins may be sufficient to provide the necessary rep functions.
  • the AAV rep and cap sequences are obtained from an AAV source as identified above.
  • the AAV rep and cap sequences may be introduced into the host cell in any manner known to one in the art as described above, including, without limitation, transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • the rep and cap sequences may be transfected into the host cell by one or more nucleic acid molecules and exist stably in the cell as an episome.
  • the rep and cap sequences are stably integrated into the genome of the cell.
  • Another embodiment has the rep and cap sequences transiently expressed in the host cell.
  • a useful nucleic acid molecule for such transfection comprises, from 5′ to 3′, a promoter, an optional spacer interposed between the promoter and the start site of the rep gene sequence, an AAV rep gene sequence, and an AAV cap gene sequence.
  • the rep and cap sequences may be supplied on a single vector, or each sequence may be supplied on its own vector.
  • the rep and cap sequences are supplied on the same vector.
  • the rep and cap sequences may be supplied on a vector that contains other DNA sequences that are to be introduced into the host cells.
  • the promoter used in this construct may be any suitable constitutive, inducible or native promoters known to one of skill in the art.
  • the molecule providing the rep and cap proteins may be in any form which transfers these components to the host cell. Desirably, this molecule is in the form of a plasmid, which may contain other non-viral sequences, such as those for marker genes.
  • This molecule does not contain the AAV ITRs and generally does not contain the AAV packaging sequences. To avoid the occurrence of homologous recombination, other virus sequences, particularly those of adenovirus, are avoided in this plasmid.
  • This plasmid is desirably constructed so that it may be stably transfected into a cell.
  • the molecule providing rep and cap may be transiently transfected into the host cell
  • the host cell be stably transformed with sequences necessary to express functional rep/cap proteins in the host cell, e.g., as an episome or by integration into the chromosome of the host cell.
  • the rep/cap proteins may be transiently expressed (e.g., through use of an inducible promoter).
  • the methods employed for constructing embodiments of this invention are conventional genetic engineering or recombinant engineering techniques such as those described in the references above.
  • the rAAV may be produced utilizing a triple transfection method using either the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif.), according to manufacturer's instructions.
  • the rAAV virions are then produced by culturing a host cell containing a rAAV virus as described herein which contains a rAAV construct to be packaged into a rAAV virion, an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression thereof.
  • Suitable viral helper genes e.g., adenovirus E2A, E4Orf6 and VA, among other possible helper genes, may be provided to the culture in a variety of ways known to the art, preferably on a separate plasmid.
  • the recombinant AAV virion which directs expression of the transgene is isolated from the cell or cell culture in the absence of contaminating helper virus or wildtype AAV.
  • RNA expression may be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and the level of protein expression may be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or by the specific methods detailed below in the examples.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • a suitable recombinant vector for use in this invention is AAV-RPE65, which utilizes AAV serotype 2 ITR and capsid sequences and is described in detail in Example 1 below.
  • This recombinant AAV contains a CMV immediate early enhancer/chicken ⁇ -actin (C ⁇ A) promoter-exon 1-intron 1 element followed by a poliovirus internal ribosome entry sequence (IRES), followed by the cDNA encoding the wildtype protein RPE65.
  • C ⁇ A CMV immediate early enhancer/chicken ⁇ -actin
  • IRES poliovirus internal ribosome entry sequence
  • the present invention is not limited to this exemplary embodiment. Similar rAAV with different transgenes, promoters, IRES, and virus capsids may be useful in this invention, as described in detail above.
  • the recombinant AAV containing the desired transgene and cell-specific promoter for use in the target ocular cell as detailed above is preferably assessed for contamination by conventional methods and then formulated into a pharmaceutical composition intended for subretinal injection.
  • a pharmaceutically and/or physiologically acceptable vehicle or carrier particularly one suitable for subretinal injection, such as buffered saline or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels.
  • a pharmaceutically and/or physiologically acceptable vehicle or carrier particularly one suitable for subretinal injection, such as buffered saline or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels.
  • buffered saline or other buffers e.g., HEPES
  • a variety of such known carriers are provided in International Patent Publication No. WO00/15822, incorporated herein by reference. If the virus is to be stored long-term, it may be frozen in the presence of glycerol.
  • the pharmaceutical composition described above is administered to the subject having such a blinding disease by subretinal injection.
  • subretinal injection as the route of delivery is a critical component of this method, as intravitreal administration does not enable the same therapeutic effects.
  • the vector and carrier cannot diffuse across the multiple cell layers in the retina to reach the RPE, when intravitreal injection is used.
  • intravenous delivery is unacceptable because the material does not penetrate the blood-brain (blood-retinal) barrier. Because the virus does not diffuse well, topical administration is similarly not preferred for this method. See the examples below.
  • An effective amount of a recombinant adeno-associated virus carrying a nucleic acid sequence encoding the desired transgene under the control of the cell-specific promoter sequence desirably ranges between about 1 ⁇ 10 9 to 2 ⁇ 10 12 rAAV infectious units in a volume of between about 150 to about 800 ⁇ l.
  • the rAAV infectious units are measured as described in S. K. McLaughlin et al, 1988 J Virol ., 62:1963. More desirably, an effective amount between about 1 ⁇ 10 10 to 2 ⁇ 10 11 rAAV infectious units in a volume of between about 250 to about 500 ⁇ l. Still other dosages in these ranges may be selected by the attending physician, taking into account the physical state of the subject, preferably human, being treated, the age of the subject, the particular ocular disorder and the degree to which the disorder, if progressive, has developed.
  • booster dosages of the pharmaceutical compositions of this invention may also be desirable to administer multiple “booster” dosages of the pharmaceutical compositions of this invention. For example, depending upon the duration of the transgene within the ocular target cell, one may delivered booster dosages at 6 month intervals, or yearly following the first administration. The fact that AAV-neutralizing antibodies were not generated by administration of the rAAV vector, as discussed in the examples below, should allow additional booster administrations.
  • Such booster dosages and the need therefor can be monitored by the attending physicians, using, for example, the retinal and visual function tests and the visual behavior tests described in the examples below. Other similar tests may be used to determine the status of the treated subject over time. Selection of the appropriate tests may be made by the attending physician. Still alternatively, the method of this invention may also involve injection of a larger volume of virus-containing solution in a single or multiple infection to allow levels of visual function close to those found in wildtype retinas.
  • an exemplary rAAVRPE65 was employed in in vitro and in vivo experiments to provide evidence of the utility and efficacy of the methods and compositions of this invention.
  • the in vitro examples demonstrated proper expression of the transgene in an animal model of a human ocular disorder resulting in blindness.
  • the in vivo examples demonstrated restoration of visual function and visual behavior by the method of this invention in a large animal model of a human retinopathy.
  • the inventors believe that although the rAAV virus targets photoreceptors and other retinal neurons as well as RPE cells, and the CPA promoter is active in all these cell types, it is likely that it is the expression of the wildtype transgene in RPE cells (and not photoreceptors) that rescues the mutant phenotype.
  • the RPE alone is responsible for, and possesses the components necessary to supply chromophore for, rod photoreceptors, although the existence of a retinal retinoid metabolism for cones (not involving RPE65 gene product) remains plausible.
  • Recombinant AAV vector was based on pTR-UF2, a vector using the 472 bp mouse rod opsin promoter to drive expression of green fluorescent protein (GFP) (Flannery, J., et al. 1997 Proc Natl Acad Sci USA 94, 6916-6921).
  • GFP green fluorescent protein
  • AAV-RPE65 the opsin promoter in pTR-UF2 was replaced with a CMV immediate early enhancer (381 bp)/chicken ⁇ -actin (C ⁇ A) promoter-exon 1-intron 1 (1352 bp) element followed by a poliovirus internal ribosome entry sequence (637 bp).
  • the latter supports expression in photoreceptors, RPE and ganglion cells (Li and Hauswirth, unpublished data, 2000).
  • the reporter/transgene GFP was replaced with the canine RPE65 cDNA (Aguirre, G. et al, 1998 Mol. Vis . 4: 23) via flanking Not I sites and the orientation and reading frame confirmed by DNA sequence analysis.
  • Plasmid DNA containing this construct was packaged into AAV particles employing iodixanol gradient purification followed by heparin-sepharose agarose column chromatography as described in Hauswirth, W. W. et al, 2000 Meth. Enzymol . 316, 743-761.
  • Vector titers were determined using an infectious center assay.
  • AAV-RPE65 virus preparations were made and combined to a total volume of 1.05 ml at 2.3 ⁇ 10 11 infectious particles/ml. Contaminating helper adenovirus and wild-type AAV, assayed by serial dilution cytopathic effect or infectious center assay, respectively were less than six orders of magnitude lower than vector AAV.
  • Example 2 in Vitro Testing of an AAV Carrying the Wildtype Canine RPE65 cDNA
  • RPE cells from eyes of both a wildtype dog and a homozygous affected (RPE65 mutant) dog were dissociated with 0.25% trypsin (Ray, J. et al, 1997 Curr. Eye Res . 16: 131-143) and plated at 1-2 ⁇ 10 5 /9 mm plastic dish. The cells were then cultured. After 48 days, confluent RPE cultures were trypsinized, subcultured and infected at 80% confluency with 2.3 ⁇ 10 7 AAV-RPE65 viral particles for 4 hours. Expression of the RPE65 transgene was assessed by immunohistochemistry 10 days post-infection.
  • the cultured canine RPE cells were evaluated by immunocytochemistry by immunolabelling with a rabbit anti-RPE65 peptide polyclonal antibody (generously provided by T. M. Redmond) and the nuclei were stained with propidium iodide.
  • a rabbit anti-RPE65 peptide polyclonal antibody generously provided by T. M. Redmond
  • proteins from cultured RPE were electrophoresed on 12.5% SDS-polyacrylamide gel and then electrotransferred on nitrocellulose membrane. Immunodetection was performed using the anti-RPE65 antibody followed by goat anti-rabbit secondary antibody and 125 I-protein A (Verdugo, M., et al. 1998 Invest Ophthalmol Vis Sci 39, S719).
  • PCR studies took advantage of the difference in size of the wildtype versus mutant canine RPE65 transcripts due to the 4 bp deletion in the latter.
  • PCR amplification utilized RPE65-1 (forward) and RPE65-3 (reverse) primers flanking the RPE65 mutant deletion site (Aguirre, G. et al, 1998 Mol. Vis . 4:23).
  • PCR conditions were 94° C. (30 seconds), 60° C. (30 seconds), and 72° C. (1 minute) for 34 cycles.
  • PCR products were separated on a 6% polyacrylamide gel.
  • AAV-RPE65 was used as positive control. This protocol was also used for PCR screening for shedding virus.
  • the PCR primers flanking this region amplified the wildtype 109 bp RPE65 DNA fragment in transduced RPE cells from an affected dog.
  • Non-transduced RPE from the same animal yielded only mutant DNA (105 bp) and normal RPE yielded only the wildtype allele (109 bp).
  • Expression of wildtype RPE65 in transduced RPE cells from an affected animal was also apparent by Western blot analysis of cell lysates. RPE65 expression was detected only in the transduced RPE cells; not in uninfected cells.
  • Each 150-200 ⁇ l subretinal injection of vector created a retinal detachment elevating approximately 35% of the total retinal area.
  • this detachment primarily occupied the nasal-inferior quadrant; in the 3rd eye (BR29) the site was temporal-superior.
  • These detachments resolved spontaneously within 24 hours. Animals were evaluated post-operatively for evidence of ocular or systemic toxicity, virus exposure to extralocular tissue, virus shedding, unfavorable immune response or other untoward effects. As discussed in detail below, none was found.
  • RPE65 expression in neural retina, RPE/choroid, and cultured RPE cells were detected.
  • Genomic PCR demonstrates persistence of transferred viral DNA in neural retina and RPE-choroid from the injected temporal-superior quadrant. In other quadrants, the host DNA amplified preferentially and the viral DNA amplification product is below detectable levels. From noninfected RPE of the affected dog, only mutant product amplifies, but 10 days posttransfection in vitro the normal transgene yields the overwhelming product.
  • RT-PCR demonstrated expression of wildtype message in neural retina from all 4 quadrants, but in RPE-choroid from the injected quadrant only. Where both products amplify, additional bands representing heteroduplexes are also seen.
  • the transfected RPE/choroid from the injected quadrant expressed higher levels of the transferred cDNA than of the mutant host gene. This was not so in other quadrants.
  • transfection of neural retina led to expression of the wildtype allele in all quadrants, a gradient was present in the relative intensities of the two alleles among quadrants.
  • the wildtype allele yielded a much more intense band than the host mutant allele. From the quadrant below this, the two bands were approximately equal in intensity. In the nasal half of the eye, the mutant band predominated.
  • ERGs Full field ERGs were recorded using a computer-based system (EPIC-XL, LKC Technologies, Inc., Gaithersburg, Md.) and Burian-Allen contact lens electrodes (Hansen Ophthalmics, Iowa city, Iowa) (Banin, E., et al. 1999 Neuron 23, 549-57). Dark-adapted luminance-response functions were obtained with blue (Wratten 47A) flash stimuli spanning ⁇ 6 log units ( ⁇ 2.9 to +2.8 log scot-cd.s.m ⁇ 2 ).
  • ERG b-wave amplitudes were measured conventionally from baseline or a-wave trough to positive peak; a-wave amplitude was measured from baseline to negative peak at the maximal stimulus.
  • dogs were light-adapted and 29 Hz flicker ERGs evoked with white flash stimuli (0.4 log cd.s.m ⁇ 2 ) on a background (0.8 log cd.m ⁇ 2 ); amplitudes were measured between successive negative and positive peaks and timing from stimulus to the next positive peak.
  • Ocular axial length and pupil diameter were measured for each experiment to permit calculation of retinal illuminance.
  • Retinal function was dramatically improved in eyes treated with subretinal AAV-RPE65, compared to pre-treatment recordings.
  • the mutant dog showed a vastly improved b-wave threshold, a large increase of a- and b-wave amplitudes (although not to normal levels) and an ERG waveform shape that is similar to controls.
  • Responses from the right eye of BR33 had b-wave thresholds lower by ⁇ 4 log units than pre-treatment, and appeared similar to normal.
  • Flicker ERGs in the same eyes as described in the immediately preceding paragraphs demonstrated a lack of detectable cone-mediated responses from RPE65 mutant dogs with untreated or intravitreally treated eyes. All eyes with subretinal AAV-RPE65 treatment recovered cone flicker responses. Cone flicker ERGs were readily recordable post-treatment; amplitudes ranged from 4 to 6 ⁇ V, representing ⁇ 16% of normal (30 ⁇ 8 ⁇ V). Intravitreally injected eyes showed no difference from untreated eyes for all measured ERG parameters.
  • a change in pupil diameter was noted in response to 2.5 log cd.m ⁇ 2 green stimulus in one eye of three representative dogs; untreated (BR46), subretinal AAV treated (BR33) and a normal control.
  • dog BR33 was consistently (5/5 observers) scored as “normally sighted” under photopic (room lighting) conditions. Under dim red light this dog consistently avoided objects either directly in front of her, or to her right (the side injected subretinally), but consistently failed to avoid objects on the left (injected intravitreally). In contrast, the untreated control affected dog, BR46 walked into objects ahead of her and at either side.
  • Table 1 provides the data collected from the procedures performed on the eyes of four RPE65 mutant dogs.
  • age is recorded as days postnatal.
  • the abbreviation Rt is used for right eye, while left is indicated for left eye.
  • the routes of injection are identified as SR for subretinal injection, IV for intravitreal injection, and NI for not injected.
  • the doses are reported as No. ⁇ 10 10 infectious particles of recombinant AAV-RPE65 virus injected.
  • Baseline ERGs were recorded 2 weeks prior to injection. Rescue Effect was assessed by ERGs recorded 95 days after injection. Positive effect is indicated by POS. NEG indicates no effect apparent.

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US12/253,955 US20090074723A1 (en) 2001-04-13 2008-10-18 Method of Treating or Retarding the Development of Blindness
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US13/766,267 US20130216500A1 (en) 2001-04-13 2013-02-13 Method of treating or retarding the development of blindness
US14/310,015 US9433688B2 (en) 2001-04-13 2014-06-20 Method of treating or retarding the development of blindness
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US12/832,282 Expired - Fee Related US8147823B2 (en) 2001-04-13 2010-07-08 Method of treating or retarding the development of blindness
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US20200172929A1 (en) * 2017-06-14 2020-06-04 The Trustees Of The University Of Pennsylvania Gene therapy for ocular disorders
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US20120225930A1 (en) 2012-09-06
US20170014529A1 (en) 2017-01-19
US8147823B2 (en) 2012-04-03
EP1381276A2 (fr) 2004-01-21
US20090074723A1 (en) 2009-03-19
US20140377224A1 (en) 2014-12-25
WO2002082904A3 (fr) 2002-12-19
US20160263246A1 (en) 2016-09-15
WO2002082904A2 (fr) 2002-10-24
EP1381276A4 (fr) 2005-02-02
US20130216500A1 (en) 2013-08-22
CA2442670A1 (fr) 2002-10-24
US20100272688A1 (en) 2010-10-28
US20070077228A1 (en) 2007-04-05

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