EP1833975A2 - Expression regulee de transgenes dans le systeme nerveux central de mammiferes - Google Patents

Expression regulee de transgenes dans le systeme nerveux central de mammiferes

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Publication number
EP1833975A2
EP1833975A2 EP05849656A EP05849656A EP1833975A2 EP 1833975 A2 EP1833975 A2 EP 1833975A2 EP 05849656 A EP05849656 A EP 05849656A EP 05849656 A EP05849656 A EP 05849656A EP 1833975 A2 EP1833975 A2 EP 1833975A2
Authority
EP
European Patent Office
Prior art keywords
rapamycin
vector
expression
transgene
aav
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05849656A
Other languages
German (de)
English (en)
Inventor
Laura Mcgee Sanftner
Victor M. Rivera
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genzyme Corp
Original Assignee
Genzyme Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genzyme Corp filed Critical Genzyme Corp
Publication of EP1833975A2 publication Critical patent/EP1833975A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention relates to regulation of genes introduced by gene therapy, specifically regulation of the expression of transgenes transduced into the central nervous system (CNS) of mammals.
  • CNS central nervous system
  • genes are under-expressed, or when the gene product itself is defective, the absence of the corresponding functional gene product may be treated by delivery of the missing gene product to the subject. Delivery of proteins, however, is often difficult and confers benefit for only a limited time, meaning that protein must be re-administered repeatedly on a regular basis, perhaps indefinitely, as in the case of chronic illnesses. Repeated administration can be expensive, inconvenient, and may suffer due to poor patient compliance.
  • the level of gene product may vary dramatically between the time just after administration of one dose and the time just before the administration of the next dose. This variability in level is particularly problematic for therapeutic agents with poor pharmacokinetics (e.g. a short half- life) or a low therapeutic index.
  • Gene therapy can be used to deliver a gene, rather than the gene product, to a cell exhibiting suboptimal expression of that gene.
  • gene therapy can also be used to deliver other genes that may have a beneficial effect when expressed in a target cell, such as cytokines, hormones, antibodies or genetically modified proteins.
  • the gene delivered by gene therapy is referred to herein as a transgene.
  • transgene When a transgene is stably expressed within the target cell, gene therapy has the potential to deliver a steady level of a gene product indefinitely. Gene therapy need only be performed once, or at least infrequently, compared to the repeated delivery of
  • Gene therapy is particularly preferred as a means for delivering
  • Viral vectors have been developed to assist in efficient delivery of transgenes to target
  • transduction a process referred to herein as transduction.
  • AAV adeno-associated virus
  • AAV Dependovirus
  • AAV dividing cells makes AAV a particularly good choice for transduction of CNS tissue, e.g.
  • AAV has not been
  • AAV is stable at a wide range of physical
  • the AAV genome is a linear, single-stranded DNA molecule approximately 4681
  • the AAV genome generally comprises an internal non-repeating genome flanked on each end by inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • the ITRs act as origins of DNA replication and as packaging
  • the internal non-repeated portion of the genome includes two main open reading
  • rep and cap genes code for the AAV replication (rep) and capsid (cap) genes.
  • the rep and cap genes code for the AAV replication (rep) and capsid (cap) genes.
  • a family of at least four viral proteins is expressed from the AAV rep region; Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weight.
  • AAV cap region encodes at least three proteins; VPl, VP2, and VP3.
  • AAV is a helper-dependent virus; that is, it generally requires co-infection with a
  • helper virus e.g., adenovirus, herpesvirus or vaccinia
  • helper virus in order to form AAV virions, hi the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral
  • helper virus infect cells from different species, the helper virus must be of the same species as the host
  • human AAV will replicate in canine cells co-infected with a canine
  • AAV vectors have been engineered to deliver genes of interest by deleting the internal
  • non-repeating portion of the AAV genome i.e., rep and cap
  • transgene between the ITRs.
  • the transgene may be linked to a heterologous
  • Termination signals such as polyadenylation sites, can also be included.
  • a suitable producer cell line is transfected with an AAV expression vector containing a
  • Helper functions are those functions normally
  • AAV-encoded genes e.g. rep and cap
  • accessory functions are those functions normally provided by helper- virus when wild-type AAV (wtAAV) replicates in
  • helper virus e.g. adenovirus
  • wtAAV must be supplied separately because they are removed in construction of rAAV
  • rAAV recombinant AAV virion
  • the rAAV stock so prepared can then be used to introduce the transgene into target
  • helper virus Because the subject's cells lack the rep and cap genes and the accessory
  • rAAV vectors are replication defective in the target cell; that is, they cannot
  • wtAAV cannot be formed in the subject's cells.
  • One such system based on induction by rapamycin (referred to herein as the
  • dimerizer system involves formation of a functional transcription factor from two
  • Rapamycin is an orally bioavailable small-molecule drug, closely related to
  • the dimerizer system has been adapted for use with viral vectors to
  • the dimerizer system is also a component of the ARGENT Transcription Technology platform of
  • the dimerizer system has the advantage of being fully humanized, in that the
  • Parkinson's disease is an example of a disease that may be amenable to gene
  • PD is the second most common neurodegenerative segment of the central nervous system.
  • dopaminergic neurons in the substantia nigra of the basal ganglia region of the brain.
  • Dopamine is synthesized in the terminal nerve endings of the dopaminergic neurons
  • nerves project into the corpus striatum, specifically innervating the putamen and the caudate
  • TH hydroxylase
  • GCH guanosine triphosphate cyclohydrolase I
  • aromatic L-amino amino acid hydroxylase
  • AADC acid decarboxylase
  • L-dopa L-dihydroxyphenylalanine
  • AADC removes the terminal carboxyl group of L-dopa to produce dopamine.
  • Treatment of PD currently involves oral administration of L-dopa, often in
  • dopa are required for therapeutic efficacy, but this often results in increased side effects.
  • One gene therapy-based approach to treatment of PD is to supply genes encoding one
  • AADC enzymes involved in dopamine biosynthesis
  • AADC restores the effectiveness of treatment of L-dopa.
  • AAV-derived vectors and methods of treatment of PD by delivery and expression of AADC in the brain of mammalian subjects are described in U.S. Pat. App. Publication No. 2002/0172664, the disclosure of which is
  • Parkinson's disease is characterized by the progressive loss of dopaminergic
  • Another gene therapy-based approach to treatment of PD is to deliver genes that block
  • GDNF GDNF
  • AAV-derived vectors and methods of treatment of PD by delivery and expression of GDNF in the brain of mammalian subjects are described in U.S. Pat. App. Publication No.
  • transgene expression be regulated to desirable levels within each transfected cell
  • dopamine can in principle be controlled by exogenous delivery of its precursor L-dopa,
  • regulation maybe beneficial to accurately control the dose of enzyme delivered, or to effect
  • neurotrophic factors such as GDNF
  • over-expression could have deleterious effects
  • CNS e.g. the brain
  • vectors enabling such regulation.
  • the regulatory system preferably, the regulatory system
  • the optimal system would also comprise functional
  • the present invention provides vectors, methods and kits for AAV-mediated gene therapy in which expression of the
  • transgene within the target cells in the nervous system can be regulated using an inducer.
  • the invention relates to recombinant AAV (rAAV) vectors in which
  • transgene expression can be regulated after the transgene has been transduced into the CNS
  • regulation is effected using a transcription factor comprising two
  • polypeptide components said transcription factor only being active when the two components
  • the two polypeptides comprise a DNA binding domain fusion
  • An exemplary DNA binding domain fusion comprises two DNA binding domains from the human transcription factor Zif268, a homeodomain derived
  • exemplary activation domain fusion comprises the rapamycin binding domain of human
  • FRAP fused to the transcriptional activation domain derived from the p65 subunit of NFKB.
  • a first rAAV vector comprises the transgene and a second rAAV
  • vector comprises the sequence of the transcription factor components.
  • regulation is accomplished by administration of an inducer, hi
  • the inducer is a dimerizer, such as rapamycin or a non-
  • immunosuppressive analog thereof e.g. AP21967.
  • the invention relates to methods of treatment of subjects with rAAV
  • transgene expression can be regulated after the transgene has been
  • treatment involves administration of an inducer, for example a dimerizer, such as rapamycin
  • a non-immunosuppressive analog thereof e.g. AP21967.
  • the invention relates to kits for constructing the vectors, or
  • the kit comprises a first rAAV
  • the kit further comprises a second rAAV vector encoding a transcription factor that can regulate expression from the transgene cloned into the first rAAV vector.
  • the kit comprises an inducer, for example a dimerizer such as
  • rapamycin or a non-immunosuppressive analog thereof e.g. AP21967.
  • the invention involves treatment of a human neurodegenerative
  • Parkinson's disease PD
  • the regulated transgene is AADC or GDNF, although in
  • the regulated transgene can be any gene whose expression in the target tissue is
  • FIG. IA is a diagram of a rAAV vector encoding the activation and DNA binding
  • AAV-CMV-TF a transcription factor for regulation of transgene expression
  • transcription factor vectors Such vectors are referred to herein as transcription factor vectors.
  • FIG. IB is a diagram of a recombinant AAV vector encoding hAADC (AAV-Z12-
  • hAADC hAADC
  • Such vectors are referred to herein as expression vectors.
  • FIG. 1C is a diagram of a recombinant AAV vector encoding hAADC (AAV-CMV- hAADC2), expression of which is driven by the constitutive CMV promoter.
  • FIG. 2 shows the results of experiments in which D7-4 cells are transduced with
  • OD values refer to the relative AADC expression (as measured by OD 4 O 5 in an AADC expression ELISA) calculated using a
  • FIG. 3 is a timeline for experiments in which the rotational response to L-dopa is
  • FIG. 4 shows the rotational response to 5 mg/kg L-dopa in 6-OHDA lesioned rats
  • Rats are infused with either excipient alone (control) or with vectors encoding regulatable hAADC and the corresponding transcription factors. Data are not presented for
  • FIG. 3 are represented as a single arrow in FIG. 4.
  • FIG. 5 A shows the results of immunohistochemistry for AADC within the striatum of
  • the scale bar represents 75 ⁇ m.
  • FIG. 5B show results of an experiment similar to that shown in FIG. 5 A but in which the rat is not treated with rapamycin.
  • FIG. 6 shows the results of immunohistochemistry for AADC in whole mounted
  • A vector-infused (+) rap
  • B vector-infused (-) rap
  • C excipient-infused (+) rap.
  • rats were infused with either excipient alone (FIG. 6C), or with vectors
  • FIGS. 6A and 6C Animals in FIGS. 6A and 6C were subsequently treated with rapamycin (as shown in FIG. 3)
  • the left hemisphere is the site of both 6-OHDA lesion and intrastriatal vector (or excipient) infusions, and the right hemisphere shows endogenous
  • FIG. 7 shows the expression of AADC at 7 weeks post-infusion, as measured by
  • top panels are images of the AADC and ⁇ -actin bands observed in gel electrophoresis of
  • the bar graph shows the average integrated image intensities
  • FIG. 8A is a diagram of an rAAV vector (AAV-TF-Z8-hGDNF) comprising a
  • hGDNF expression can be regulated by addition of rapamycin or derivatives thereof.
  • FIG. 8B is a diagram of a vector similar to that shown in FIG. 8 A, except that
  • FIG. 8C is a diagram of a recombinant AAV vector encoding hGDNF, expression of
  • FIG. 9 shows GDNF expression, in picograms (pg), for HeLa D7-4 cells transiently
  • a regulated TF-GDNF plasmid AAV-TF-Z8-hGDNF
  • a regulated TF-GDNF plasmid AAV-TF-Z8-hGDNF
  • a vector includes a mixture of two or more such vectors, and the like.
  • transgene refers to any gene to be delivered to a target cell
  • Transgenes may direct production of messenger RNAs encoding proteins, or they may encode biologically active RNA molecules, such as antisense, ribozyme, triplex-forming,
  • RNAi or other RNA sequences are examples of RNA sequences.
  • a "regulatable transgene,” as used herein, is a gene whose expression can be altered
  • transgene expression provides pharmacologic control of the level of transgene
  • “Expression cassette” refers to an assembly which is capable of directing the
  • the expression cassette includes a
  • promoter or promoter/enhancer which is operably linked to (so as to direct transcription of)
  • sequence(s) or gene(s) of interest and often includes a polyadenylation sequence as well.
  • adeno-associated virus construct contained within an adeno-associated virus construct.
  • Recombinant as used herein to describe a nucleic acid molecule means a
  • polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue
  • polypeptide means a polypeptide produced by expression of a recombinant polynucleotide, hi
  • the gene of interest is cloned and then expressed in transformed organisms, as
  • the host organism expresses the foreign gene to produce the protein
  • a "coding sequence” or a sequence which "encodes” a selected polypeptide is a
  • nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of
  • sequences (or "control elements").
  • the boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy)
  • a coding sequence can include, but is not limited to, cDNA from viral, procaryotic
  • a transcription termination sequence may be located 3' to the
  • control elements include, but are not limited to, transcription promoters,
  • transcription enhancer elements transcription termination signals, polyadenylation sequences
  • nucleic acid includes DNA and RNA, and also their analogues, such as
  • PNA nucleic acids
  • transfection is used to refer to the uptake of foreign DNA by a cell.
  • a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
  • DNA moieties such as a plasmid vector and other nucleic acids
  • transduction denotes the delivery of a DNA molecule to a recipient cell either in vivo or in vitro, via a vector, such as a recombinant adeno-associated virus vector.
  • a vector such as a recombinant adeno-associated virus vector.
  • heterologous as it relates to nucleic acid sequences such as gene sequences
  • control sequences denotes sequences that are not normally joined together, and/or are not
  • nucleic acid normally associated with a particular cell.
  • a heterologous region of a nucleic acid normally associated with a particular cell.
  • construct or a vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature.
  • a nucleic acid molecule that is not found in association with the other molecule in nature.
  • heterologous region of a nucleic acid construct could include a coding sequence flanked by
  • heterologous coding sequence is a construct where the coding sequence itself is not found in
  • control elements refers collectively to promoter regions, polyadenylation
  • IVS internal ribosome entry sites
  • enhancers enhancers
  • sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • promoter region is used herein in its ordinary sense to refer to a nucleotide
  • RNA polymerase from a gene which is capable of binding RNA polymerase and initiating transcription of a
  • “Operably linked” refers to an arrangement of elements wherein the components so
  • control elements operably linked to a coding sequence are capable of effecting the expression of the coding
  • control elements need not be contiguous with the coding sequence, so long as
  • transcribed sequences can be present between a promoter sequence and the coding sequence
  • promoter sequence can still be considered "operably linked" to the coding sequence.
  • isolated when referring to a nucleotide sequence, is meant that the indicated
  • an "isolated nucleic acid molecule which encodes a particular polypeptide” refers
  • nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases
  • a "vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral
  • vectors typically, vector constructs, non-viral vectors, particulate carriers, and liposomes.
  • vector construct typically, vector construct
  • expression vector means any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid
  • sequences to target cells include cloning and expression vehicles, as well as
  • subject any member of the subphylum chordata, including, without
  • humans and other primates including non-human primates such as chimpanzees
  • farm animals such as cattle, sheep, pigs, goats and
  • mice as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as
  • regulatable transcription factor and/or transgene is intended an amount that when the AAV
  • ameliorates symptoms or prevents progression of a neurological disorder ameliorates symptoms or prevents progression of a neurological disorder.
  • a therapeutic agent e.g., AADC, GDNF
  • Parkinson's disease may ameliorate symptoms, e.g., improve motor function or reduce
  • the present invention provides vector constructs, methods and kits for regulation of
  • transduction of a regulatable transgene in a target cell are provided on a plurality of separate
  • Examples 1 and 2 demonstrate the construction and use of a dual-vector embodiment of the present invention. In another embodiment, a single
  • recombinant AAV vector carries all the sequences necessary for transduction of a regulatable
  • Example 3 demonstrates the construction and use of one such
  • a regulatable form of the AADC gene is delivered to target cells.
  • Examples 1 and 2 demonstrate delivery of a regulatable form of the AADC gene, hi another
  • a regulatable form of the GDNF gene is delivered to target cells.
  • transgene refers to any gene to be delivered to a target cell
  • Transgenes may direct production of messenger RNAs encoding proteins, or they
  • RNA molecules may encode biologically active RNA molecules, such as antisense, ribozyme, triplex-forming,
  • a regulatable transgene is a gene whose expression can be altered by
  • transgene expression provides pharmacologic control of the level of transgene
  • the inducer is a small molecule drug.
  • molecule drug is rapamycin.
  • the regulatable gene expression system of the present invention is operable to control the expression of the present invention
  • rapamycin analog to produce an active transcription factor complex that specifically activates
  • AP21967 (molecular mass 1017.4 Da) is the 7-methylindolyl analog of Compound 69
  • AP21967 is identical to Compound 71 of the '595 patent except that AP21967 has a 7-methyl group on the indole ring.
  • Rapamycin analogs that do not interact with the endogenous FRAP in the
  • FRAP forms of FRAP can be created that retain the ability to bind to these non-immunosuppressive
  • rapamycin analogs for use in the FRB portion of the activation domain fusion protein of the
  • dimerizer system See, e.g., Pollock et al. (2000).
  • transgene is made dependent on reconstitution of a functional transcription factor (TF) by the
  • transcription factor AAV vector is used to deliver the DNA binding domain and the activation
  • an expression AAV vector is used to deliver the
  • hAADC transgene under the control of a regulatable promoter.
  • Example 1 The dimerizer used in Example 1, AP21967, is a non-reacted dimerizer
  • promoter at 25 nM AP21967 is roughly half the level from hAADC under control of the
  • AAV-CMV-hAADC2 constitutive CMV promoter (AAV-CMV-hAADC2) (FIG. 1C, and described at Sanftner et al.
  • OHDA OHDA rat model of Parkinson's disease
  • a surgical model of striatal denervation as
  • Parkinsonian rats are transduced with both AAV-CMV-TF and AAV-Z 12-hAADC and their
  • Rapamycin is used in the in vivo experiments, rather than AP21967 as was used in
  • the dimerizer rapamycin has many favorable properties for use as an inducer in human gene
  • immunosuppressive analogs of rapamycin such as AP21967, may be superior inducers of
  • transgene expression particularly in treatment of human subjects.
  • AP21967 in animals and human subjects in vivo may be determined by standard experimental
  • L-dopa and rapamycin results in behavioral effects in 6-OHDA-lesioned rats consistent with production of significant levels of dopamine. As illustrated in FIG. 4, treatment with
  • rapamycin reversibly increases hAADC expression in the lesioned striatum, as evidenced by
  • FIG. 5A demonstrates strong immunohistochemical staining for AADC, and thus
  • FIG. 5B shows only very low level AADC
  • FIGS. 6A-6C present low resolution images of whole mounted brain sections
  • Table 1 presents the results of transgene-derived immunostaining
  • Rapamycin-induced animals show approximately twice the anterior-to-
  • FIG. 7 Western blot analysis of hAADC enzyme levels after gel electrophoresis of striatal
  • induction is not enough to elicit a behavioral response to a sub-therapeutic dose of L-dopa
  • induction in vivo may be determined by experimentation on a case-by-case basis, as is
  • Dosage may be adjusted by trial and error based on
  • exemplary dosages might range from
  • 0.01 to 50 mg/kg preferably 0.1 to 10 mg/kg, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 0.9,
  • Recombinant AAV vectors can be surgically introduced at various locations in the
  • the basal ganglia are groups of neurons positioned subcortically.
  • the caudate nucleus and the caudate nucleus include the caudate nucleus, putamen, and globus pallidus.
  • the caudate nucleus and the caudate nucleus include the caudate nucleus, putamen, and globus pallidus.
  • putamen together form the corpus striatum (or simply striatum).
  • the caudate and putamen are reciprocally interconnected with the substantia nigra, which consists of the substantia
  • SNpc nigra pars compacta
  • SNpc nigra pars compacta
  • dopamine synthesis can be any enzymes in the dopamine biosynthetic pathway, such as AADC, dopamine synthesis.
  • Corpus striatal cells can be transduced using a variety of techniques known in the art.
  • stereotaxic injection is a common surgical technique used by neurosurgeons to create stereotaxic injection.
  • Direct injection can also be employed; if using this technique, anatomical maps derived from CT, PET, or MRI scans can be used by the
  • hAADC in the expression vector.
  • expression vectors like the one illustrated at FIG. IB e.g. ITR regions, promoter,
  • transgene sequences in the expression vector are transcription factor binding sequences, etc.
  • any desired transgene can be delivered using the rAAV expression- vector constructs of a dual- vector embodiment of the present invention.
  • shorter ITR, promoter or transcription factor binding regions may be constructed to enable
  • hAADC transgene can be regulated in human cells in culture, and in rat neurons in vivo, using
  • FIG 8 A A diagram of a regulated rAAV hGDNF expression vector is provided at FIG 8 A,
  • FIG. 8C a diagram of a control vector with constitutive (unregulated) hGDNF expression is provided as FIG. 8C.
  • FIG. 8B shows an alternative design for a regulated GDNF expression
  • Plasmid vectors are transiently transfected into HEK-
  • FIG. 9 The data show rapamycin-dose-responsive expression of GDNF in cells transfected
  • TF-GDNF plasmid which refers to pAAV-TF-Z8-hGDNF
  • CMV-GDNF plasmid the constitutively expressed GDNF
  • GDNF expression from pCMV-GDNF is not increased by addition of rapamycin.
  • Example 3 The experiment described in Example 3 demonstrates that a single- vector regulatable
  • AAV-GDNF construct can be constructed that in which GDNF expression can be regulated in human cells in culture simply by addition of the small molecule inducer rapamycin.
  • Treatment with a single vector has the advantage of requiring only one transduction
  • transcription factor fusion proteins and regulatory elements used in the rAAV vector are used in the rAAV vector.
  • transgene (GDNF) sequence is
  • transgene sequences e.g. up to 850 or
  • transgenes may comprise active sub-fragments of desirable genes,
  • rAAV virions rather than full-length genes, to facilitate single- vector delivery.
  • units of dose in vector genomes/per kilogram of body weight may depend on several
  • the proper dose of rAAV used to effect transduction in a mammal may range from
  • GDNF are presented as exemplary transgenes in the examples herein, the specific transgene to be delivered is not a limiting aspect of the present invention.
  • genes that might be expected to provide a beneficial (e.g. therapeutic) effect may be
  • sequence is short enough to fit into an AAV vector construct that can be
  • TH tyrosine hydroxylase
  • neurotrophins include neurotrophins, including GDNF (OMEVI 600837, Genbank Accession No. AX713049, L19063) and other members of the GDNF protein family, such as artemin (OMIM 603886, Genbank Accession No. AF109401), neurturin (OMM 602018,
  • transgenes include IL-IO (OMM 124092, Genbank Accession No. M57627).
  • OMIM numbers refer to the Online Mendelian Inheritance in Man database
  • Genbank accession numbers are provided for representative complete cDNA
  • transgenes include other sequences reported for the gene in Genbank, full-length and
  • Transgenes also include genes for treatment of other neurodegenerative diseases, such as Alzheimer's, diabetes, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, such as
  • Alzheimer's disease Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Canavan disease, cerebral ischemia, progressive supranuclear palsy, Lewy body
  • corticobasal degeneration multiple system atrophy and retinal degeneration (e.g. macular
  • the inducer is preferably selected from among compounds that can be delivered to a
  • the inducer is able to cross the blood-brain barrier.
  • the inducer is a dimerizer, for example rapamycin or
  • the inducer is delivered parenterally, e.g. by subcutaneous,
  • the inducer is any substance that is administered to the patient intramuscular, intraocular or intravenous injection.
  • the inducer is any substance that is administered to the patient.
  • nasal delivery nasal spray
  • aerosol/pulmonary delivery inhaler
  • ocular delivery eye drops
  • Inducer may also be delivered continuously or semi-
  • minipump a mechanical infusion pump or a controlled release pharmaceutical composition.
  • Stocks of rAAV vectors according to the present invention may be prepared using any
  • Wild-type AAV and helper viruses can be used to provide the necessary replicative functions for producing rAAV
  • helper function genes e.g. pHLP 19
  • the accessory function genes e.g. pladeno 5
  • both in the case of the triple transduction are the accessory function genes (e.g. pladeno 5), or both in the case of the triple transduction
  • rAAV virions are formulated into pharmaceutical compositions
  • excipients include any pharmaceutical agent that does not itself
  • liquids such as water, saline, glycerol and ethanol.
  • hydrochlorides such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the
  • rAAV virions of the present invention are supplied as
  • compositions comprising excipients that enhance viral stability during
  • excipients exhibit low toxicity in the
  • target tissue e.g. the CNS.
  • virions can be stored and supplied in buffer
  • kits for construction of AAV vectors are provided.
  • one or more vectors for regulated expression of transgenes.
  • vectors similar to those shown in FIGS. IB, 8A or 8B are identical to those shown in FIGS. IB, 8A or 8B.
  • FIG. IB are useful in dual- vector methods, whereas vectors similar to those shown in
  • FIGS. 8 A and 8B are useful in single- vector methods of the present invention.
  • a user of a kit of the present invention can clone a gene or gene fragment of interest into an expression
  • Kits including expression vectors for use in dual- vector methods may also include a
  • transcription factor vector substantially similar to that shown in FIG. IA.
  • Kits may optionally include rapamycin or an analog thereof.
  • Kits may also optionally include plasmids for use in preparing rAAV virion stocks,
  • Kits may also contain instructions for use.
  • gene therapy vectors, methods and kits of the present invention are administered to a subject afflicted with a disease, such as Parkinson's disease, to provide a disease.
  • a disease such as Parkinson's disease
  • therapeutic effect refers to a level of expression of one or more transgenes sufficient to alter a component of a disease (or disorder) toward a
  • a therapeutic effect can be an
  • a reduction in resting tremor can also be a
  • PPRS Primate Parkinsonism Rating Scale
  • CRS Clinical Rating Score
  • bradykinesia bradykinesia, hypokinesia, and muscular rigidity.
  • the PPRS system is described in Langston
  • FIG. IA is a diagram of AAV-CMV-TF, in which
  • CMV cytomegalovirus
  • domain fusion protein contains the rapamycin binding domain (FRB*) of human FRAP fused
  • FRB domain used in this embodiment (FRBT 2 O9SL) 3 and illustrated in FIG. IA (FRB*), carries a T to L mutation at position 2098 when compared to the wild-type FRB
  • FRB T2098L can be dimerized to the DNA binding domain fusion using either AP21967 or rapamycin.
  • the internal ribosome entry sequence (IRES) is derived from encephalomyocarditis
  • the DNA binding domain fusion protein contains two DNA binding domains from the
  • FIG. IB is a
  • ITR inverted terminal repeat
  • hAADC hAADC coding sequence
  • human growth
  • HeLa D7-4 cells with a 1:1 ratio AAV-CMV-TF and AAV-Z12-hAADC, and subsequently
  • AAV-CMV-TF transcription factor vector alone
  • hAADC vector AAV-CMV-hAADC2
  • hAADC vector (AAV-Z12-hAADC) but lacking the transcription factor vector.
  • AAV-CMV-TF has been described previously (AAV-CMV-TF INc, Auricchio et al.
  • AAV-Z12-hAADC is created by replacing the CMV enhancer
  • Recombinant AAV vectors (serotype 2) are generated by triple transfection of
  • HEK-293 ATCC Accession No. CRLl 573 cells and purified by CsCl density gradient
  • rAAV are microfluidized and filtered through 0.2- ⁇ m filters.
  • Vector is purified from the
  • PAGE gels. Titer is determined by Q-PCR analysis of vector genomes.
  • the expression ELISA measures the expression of hAADC protein in permeabilized
  • HeLa D7-4 cells using an antibody against hAADC. HeLa D7-4 cells are transduced with
  • Cells are seeded in a 96-well plate 24 hr before transduction. Cells are transduced with an MOI of 10 4 vg/cell (of each vector when multiple vectors are used) in 100
  • ELISA is performed on transduced cells 48 h post-transduction. Briefly, media are
  • the primary antibody (AB 136 rabbit anti-
  • hAADC, Chemicon, 1 :1000 is diluted in wash buffer (1% goat serum, 0.5% Triton X 100 in
  • FIGS. IA and IB are also tested in vivo in the 6-
  • hydroxydopamine (6-OHDA) rat model of Parkinson's disease as follows. Rapamycin is used instead of AP21667 as dimerizer in Example 2 because of its higher potency and known pharmacokinetics. Rapamycin has a half-life of approximately 1Oh in vivo with rapid
  • excipient-infused control group excipient-infused (+)
  • rapamycin treatment a vector-infused (-) rapamycin treatment control group, and a vector-
  • the vector-infused (-) rapamycin group serves as a control for hAADC gene expression in the absence of rapamycin, i.e. to determine whether
  • the system is leaky.
  • FIG. 3 shows the experimental timeline for the experiments described in this example.
  • Induction consists of four
  • Rats are allowed to recover from rapamycin for a rotational response to 5 mg/kg L-dopa. Rats are allowed to recover from rapamycin for
  • Rats are induced a second time on day 31
  • the agonist is dopamine synthesized from exogenous L-dopa, in the
  • rapamycin group show a robust contralateral turning response to L-dopa (5 mg/kg) that is
  • (+) rapamycin group (P ⁇ 0.001).
  • the vector-infused (+) rapamycin group is not
  • the vector-infused (-) rapamycin and the excipient-infused control groups are not significantly different at any of the time points (P > 0.05). Without intending to be bound by
  • control groups may be due to sensitization to repeated L-dopa treatment.
  • time points, weeks 4 and 6 is not significantly different from the vector-infused (-) rapamycin
  • infused groups are transduced with equivalent numbers of copies of hAADC gene.
  • hAADC expression levels are evaluated by immunohistochemical analysis at seven
  • FIG. 5 A representative animal from the vector-infused (+) rapamycin group is shown in FIG. 5 A.
  • FIG. 5B one from the vector-infused (-) rapamycin group is shown at FIG. 5B. As illustrated in
  • FIG. 5 A hAADC transgene expression is localized to the medium spiny neurons in the rat
  • FIG. 5B shows low magnification images of AADC immunohistochemistry in whole
  • the left hemisphere is the site of
  • 6-OHDA lesioning causes depletion of the endogenous AADC, resulting in low
  • Rats all exhibit hAADC transgene staining on the infused left side (see, e.g., FIG. 6A). Rats
  • rapamycin dosing is evaluated and quantitated by stereology in
  • n is the number of hemispheres examined.
  • Average anterior-to-posterior spread, volumes of spread, and positive cell numbers for the rapamycin-induced group are statistically different from the values for the "no induction" group (PO.02) by Student's t-tests.
  • Table 1 also presents the average population of transgene positive cells and the
  • the vector-infused (+) rapamycin group has the
  • hAADC created per cell may differ between groups.
  • hAADC expression in the uninduced group seen by immunostaining correlates with a lower
  • total protein is extracted from serial tissue sections and
  • ⁇ -Actin is included as a loading control.
  • AADC band density is significantly higher in the vector-
  • CED convection-enhanced delivery
  • Vector is delivered with a programmable pump (Bioanalytical Systems, hie, West Lafayette,
  • the cannula consisting of a fused silica capillary (OD, 164 ⁇ m; ID, 100 ⁇ m; Polymicro
  • the target sites and cannulas are inserted vertically into the caudate-putamen at the following
  • contralateral and ipsilateral turns are computed over 30 min (for apomorphine) and over 60
  • L-dopa response is evaluated before surgery and at different time points (3, 4, 5, 6, and 7
  • animals are perfused through the aorta with saline, followed
  • rapamycin groups and n 8 rats for the vector-infused (+) rapamycin group). Brains are
  • Brains are cut serially into 40- ⁇ m thick coronal sections on a cryostat.
  • Sections are incubated in 3% hydrogen peroxide for 30 min to quench
  • n is the number of sections with hAADC positive cells, 40 ⁇ m is the
  • the vector AAV-Z12-hAADC used in this study contains the human AADC target
  • the plasmid is linearized with a
  • hAADC gene copies is calculated by comparison to the standard curve, and multiplying the
  • Proteinase inhibitors Protein is quantified using the Bradford method. Protein samples (15
  • the filters are blocked with 3% milk and incubated for 1 hr with a polyclonal rabbit
  • the anti-AADC primary antibody has
  • Mammalian cells are transduced with a dimerizer-regulatable GDNF transgene
  • FIGS. 8 A and 8B are diagrams of recombinant AAV vector plasmid constructs for
  • hGDNF regulatable human GDNF
  • pAAV-CMV-hGDNF constitutively expressed hGDNF
  • the regulatable constructs involve a single rAAV vector carrying
  • hGDNF is driven by a minimal IL-2 promoter adjacent to eight binding sites for the dimerizable transcription factor described in greater detail below.
  • a CMV construct illustrated at FIG. 8A ⁇ AAV-TF-Z8-hGDNF
  • enhancer/promoter drives expression a single transcript encoding both the activation and
  • an SV40 promoter drives
  • the activation domain on a different transcript, from the opposite strand (i.e. in the opposite
  • the DNA binding domain fusion protein contains two DNA binding domains from
  • the activation domain fusion protein contains the rapamycin binding domain of
  • Min hGH pA hormone polyadenylation
  • control vector pAAV-CMV-hGDNF (FIG. 8C) the CMV promoter/enhancer drives
  • polyadenylation (pA) sequences are indicated.
  • GDNF expression is presented as a function of vector construct and rapamycin concentration.
  • pAAV-TF-Z8-hGDNF directs production of GDNF in a dose-responsive manner when cells are treated with rapamycin, whereas pAAV-CMV-hGDNF directs constitutive (high) level

Abstract

L'invention concerne des vecteurs du virus adéno-associé recombinant (rAAV) pour l'administration de transgènes régulables dans le système nerveux central (SNC) d'un mammifère. L'invention concerne également des méthodes de traitement de sujets présentant des troubles neurodégénératifs au moyen de ces vecteurs, ainsi que des trousses destinées à construire ou utiliser ces vecteurs ou à mettre en oeuvre lesdites méthodes. Des séquences transgéniques sont exprimées à partir d'une région promotrice/activatrice comprenant un ou plusieurs sites de liaison pour un facteur de transcription sensible à un inducteur moléculaire de petite taille. La construction transgénique et une construction comprenant le facteur de transcription sont administrées dans les cellules cibles. Le transgène régulable peut être administré sur le même vecteur rAAV que le facteur de transcription ou sur un vecteur séparé. Le facteur de transcription peut comprendre deux chaînes polypeptidiques, telles qu'un domaine de liaison à l'ADN et un domaine d'activation de transcription, qui forment un dimère actif en présence d'un agent de dimérisation tel que la rapamycine ou un analogue non immunogène correspondant. Les vecteurs, les méthodes et les trousses de l'invention peuvent être utilisés pour administrer des gènes tels que GDNF ou AADC dans le cerveau d'un sujet présentant un trouble neurodégénératif tel que la maladie de Parkinson, l'expression de GDNF ou AADC dans le cerveau pouvant ensuite être régulée par traitement du sujet avec de la rapamycine ou un analogue de rapamycine.
EP05849656A 2004-12-09 2005-12-09 Expression regulee de transgenes dans le systeme nerveux central de mammiferes Withdrawn EP1833975A2 (fr)

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WO2010138263A2 (fr) 2009-05-28 2010-12-02 University Of Massachusetts Nouveaux virus adéno-associés (aav) et leurs utilisations
CN104232686A (zh) * 2014-08-20 2014-12-24 江苏大学 小鼠维甲酸相关的孤核受体α腺相关病毒载体
US10480011B2 (en) * 2014-10-21 2019-11-19 University Of Massachusetts Recombinant AAV variants and uses thereof
CA2966620A1 (fr) 2014-11-05 2016-05-12 Voyager Therapeutics, Inc. Polynucleotides codant pour la dopa decarboxylase et destines au traitement de la maladie de parkinson
WO2017075335A1 (fr) 2015-10-28 2017-05-04 Voyager Therapeutics, Inc. Expression régulable au moyen d'un virus adéno-associé (vaa)
CA3035522A1 (fr) 2016-08-30 2018-03-08 The Regents Of The University Of California Procedes de ciblage et d'administration biomedicaux, et dispositifs et systemes pour la mise en ƒuvre de ceux-ci
EP3526333A4 (fr) 2016-10-13 2020-07-29 University of Massachusetts Conceptions de capsides de vaa
JOP20190269A1 (ar) 2017-06-15 2019-11-20 Voyager Therapeutics Inc بولي نوكليوتيدات aadc لعلاج مرض باركنسون
WO2019018342A1 (fr) 2017-07-17 2019-01-24 Voyager Therapeutics, Inc. Systeme de guide de trajectoire d'appareillage en reseau
GB201717524D0 (en) 2017-10-25 2017-12-06 Autolus Ltd Vectors

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CN101213305A (zh) 2008-07-02
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