A vector and a method are provided for delivering a nucleic acid to a nervous system cell. The vector includes a first nucleic acid, a second nucleic acid, inverted terminal repeats of adeno-associated virus, and a tetracycline-off regulatable promoter system that includes a first promoter operably linked to the first nucleic acid and a second promoter operably linked to the second nucleic acid. The promoters drive expression in opposite directions and away from the inverted terminal repeats. The method includes providing a recombinant adeno-associated viral (rAAV) vector and administering the vector to a nervous system cell. Expression of a product from the first nucleic acid is regulatable by the promoter system.

Description of the Invention

BRIEF SUMMARY

In one aspect of the present invention, a regulatable recombinant adeno-associated viral vector is provided. The vector includes a first nucleic acid for providing a therapeutic effect on a nervous system disorder when a product is produced from the first nucleic acid and a second nucleic acid encoding a tetracycline-controlled transactivator. The vector further includes inverted terminal repeats from adeno-associated virus and a tetracycline-off regulatable promoter system having a first promoter operably linked to the first nucleic acid and a second promoter operably linked to a second nucleic acid. The first and second promoters drive expression in opposite directions, towards each other and away from the inverted terminal repeats.

In another aspect of the present invention, a method of delivering a first nucleic acid to a nervous system cell in a patient having a nervous system disorder is provided. The method includes providing a recombinant adeno-associated viral (rAAV) vector and administering the vector to a nervous system cell. The vector includes the first nucleic acid encoding a protein that provides a therapeutic effect on the nervous system disorder, a second nucleic acid encoding a tetracycline-controlled transactivator, inverted terminal repeats of adeno-associated virus, and a tetracycline regulatable promoter system that includes a first promoter operably linked to the first nucleic acid and a second promoter operably linked to the second nucleic acid. Expression of a product from the first nucleic acid is regulatable by the promoter system.

In yet another aspect of the present invention, a method of making recombinant AAV virions is provided. The method includes providing a recombinant adeno-associated viral vector (rAAV) and an AAV derived helper plasmid to producer cells, providing a helper virus; and culturing the producer cells to make the virions. The rAAV vector includes the first nucleic acid, a second nucleic acid encoding a tetracycline-controlled transactivator; inverted terminal repeats of AAV; and a tetracycline-off regulatable promoter system comprising a first promoter operably linked to the first nucleic acid and a second promoter operably linked to the second nucleic acid, wherein the first and second promoters drive expression in opposite directions in the vector, towards each other and away from the inverted terminal repeats.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

The present invention will utilize a rAAV vector for delivery of therapeutic genes to the nervous system. The rAAV vector includes a regulatable promoter system for temporally regulating gene expression.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijssen, ed.); Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.) All references cited herein are incorporated by reference.

Regulatable rAAV Vector

The vector of the present invention is a recombinant adeno-associated viral vector which includes the ITRs of AAV. The vector also includes a tet regulatable promoter system, and a therapeutic nucleic acid.

In a preferred embodiment of the present invention, ITRs from AAV2 are provided in a vector and the ITRs flank the tet-regulatable promoter system and the therapeutic nucleic acid. Preferably all other AAV sequences are deleted from the rAAV vector. The ITRs may be wild-type or recombinant and the ITRs do not need to be identical as long as the ITRs allow for packaging of the virions.

The tet system may be used to regulate expression of the therapeutic nucleic acid. As discussed above, the preferred tet-regulatable system of the present invention minimizes the leaky regulation of the therapeutic nucleic acid.

To exemplify the invention, a rAAV vector, rAAVS3, is constructed as follows. One of skill in the art will understand that alternative promoters, enhancers, other regulatory elements, and therapeutic nucleic acids may be used in the construction of the tet-regulatable rAAV vector of the present invention. As shown in FIGS. 1-3 (see Original Patent), the preferred rAAVS3 vector includes a cytomegalovirus (CMV) promoter, a tetracycline-controlled activator (tTA), a minimal CMV promoter (P.sub.CMV), a tet response element (TRE), a Simian virus 40 polyadenylation signal (SV40pA), and a therapeutic nucleic acid, flanked by AAV ITRs from AAV2. In order to control regulation of the therapeutic nucleic acid, the expression cassettes for the therapeutic nucleic acid and the tTA are driven in opposite directions, away from the ITRs and towards each other. The expression cassette for the therapeutic nucleic acid includes the TRE having seven copies of the tet resistance operator binding sites in frame with the P.sub.CMV driving the therapeutic nucleic acid, followed by the SV40pA. The tTA expression cassette includes the CMV promoter driving a tet repressor (tet R) in frame with a nucleic acid encoding the C-terminal 127 amino acids of the herpes simplex virus VP16 activation domain (VP 16) and terminating in SV40pA. Tight regulation provided by the rAAVS3 vector is shown in FIGS. 5A and 5B (see Original Patent) in comparison to two vectors, rAAVS1 and rAAVS2, having the same expression cassettes, but driven in alternative directions to rAAVS3 (constructs shown in FIG. 4 (see Original Patent)). In rAAVS1, the therapeutic nucleic acid and the tTA expression cassettes are driven away from each other and toward the ITRs. In rAAVS2, the therapeutic nucleic acid and tTA expression cassettes are driven in the same direction. As will be described in the examples below, in comparison to rAAVS1 and rAAVS2 vectors, the rAAVS3 vector can be used to tightly regulate expression of the therapeutic nucleic acid, providing a non-leaky promoter system with about 10% or less, preferably with about 5% or less expression in the "OFF" state compared to the "ON" state, more preferably with about 1% or less expression in the "OFF" state compared to the "ON" state.

In some embodiments, the rAAV vector may include a Kozak-like consensus sequence to facilitate expression of the therapeutic nucleic acid. (See, for example, Kozak, M, J. Biol. Chem., 266(30): 19867-19870, 1991.) Exemplary Kozak-like consensus sequences are described in the examples below. Any Kozak-like consensus sequence may be included in the rAAV vectors of the present invention and the sequences are not limited to the examples given below.

In a preferred embodiment, the therapeutic nucleic acid encodes a product for treatment of a nervous system disorder. The product may be, by way of example, but not limited to, a protein or an RNA molecule such as an inhibitory RNA molecules including ribozymes, antisense RNAs, small inhibitory RNAs (RNAi) and microRNAs.

Suitable nucleic acids include, but are not limited to, those encoding proteins for the treatment of nervous system disorders as described above. Suitable genes for the treatment of nervous system disorders, for example, using the rAAV vector of the present invention encoding, but not limited to, glial cell-line derived neurotrophic factor (GDNF) (Genbank HUMGDNF02; Accession No. L19063), and other members of the GDNF family including neurturin, perspephin and artemin, aromatic amino-acid decarboxylase (AADC) (Genbank HUMDDC, Accession No. M76180); (brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) (EMBL HSNGF2; Accession No. X53655, and/or other members of the neurotrophin factor family including neurotrophin (NT)-3 (Genbank HUMBDNF; Accession No. M37762) and NT-4 (Genbank HUMPPNT4P; Accession No. M86528), bone morphogenetic proteins such as BMP4, ciliary neurotrophic factor (CNTF), platelet-derived neurotrophic factor (PDGF), leukemia inhibitory factor (LIF), interleukins, such as IL-2 and IL-12, tyrosine hydroxylase (TH) (Genbank HUMTHX, Accession No. M17589), dopamine-.beta.-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), tryptophan hydroxylase, vesicular monoamine transporter (VMAT), dopamine transporter (DAT) and other neuronal transporters such as catecholamine, serotonin and glutamate transporters, vascular endothelial growth factor (VGEF).

Other suitable nucleic acids include superoxide dismutase (SOD1 or SOD2) (GenBank HUMCUZNDI; Accession No. M12367; for SOD-1, EMBL HSSOD2G, Accession No. X65965 for SOD-2), catalase (EMBL HSCATR, Accession No. X04076), and/or glutathione peroxidase (MBL HSGSHPX, Accession No. Y00433); adenosine A-1 receptor (GenBank S56143; Accession S56143); glutamate decarboxylase (GenBank S61898; Accession S61898), choline acetyltransferase (ChAT), cholinergic nicotinic or muscarinic receptors, neuropeptides, including but not limited to enkephalin, dynorphin, substance P, neuropeptide Y, GABA-A receptor isoforms (EMBL HSGABAAA1; Accession X14766), calcium-dependent potassium channels (GenBank DROKCHAN, Accession M96840) and/or ATP-sensitive potassium channels (Ho, et al 1993 Nature 362:31-8). Also included are nucleic acids involved in regulation of cell death such as bcl2 and other members of the bcl2 family such as, bcl.sub.x1 and bax, and caspase enzymes and dominant-negative nucleic acids against caspase enzymes, and tumor necrosis related apoptosis-inducing factor (TRAIL). Nucleic acids encoding for molecules that stimulate or inhibit growth of neuronal processes, such as nogo, netrins, semaphorins, N-CAM, eph receptors and eph ligands, and artificial peptides that interfere with intracellular signaling mechanisms and transcription factors involved in neuronal growth and function such as sonic hedgehog (SHH), nurr-1, HOX genes, LIM genes, POU genes, c-fos and other members of the SP1 family, such as fosB are suitable for use in the present invention. The nucleic acids for use in the present invention include the deposited nucleic acids listed above as well as any nucleic acids having conservative substitutions, sufficient homology, or encoding RNA or the proteins listed above for the treatment of nervous system disorders.

The gene can code for a transmitter, such as acetylcholine or GABA, or a receptor for such a transmitter or a gene that encodes a growth factor such as FGF (fibroblast growth factor), EGF (epidermal growth factor), glial growth factor, PDGF (platelet-derived growth factor) or a cytokine, or the like.

Virion Production

rAAV virions, which include the rAAVS3 vector, can be produced using standard methodology, known to one of skill in the art. The methods generally involve the steps of introducing the rAAV vector containing the therapeutic nucleic acid and an AAV-derived helper plasmid into a producer cell, where the helper construct includes AAV coding regions capable of being expressed in the producer cell to complement AAV helper functions missing from the rAAV vector wherein AAV helper functions include, but are not limited to, one, or both of the major AAV ORFs, namely the rep and cap coding regions, or functional homologues thereof; and helper functions from herpes virus or adenovirus, such as EA2, E3 and E4; and culturing the producer cell to produce rAAV virions. The AAV expression vector, AAV helper construct(s) can be introduced into the producer cell, either simultaneously or serially, using standard transfection techniques known to one of skill in the art (Zoltukhin et al., Gene Therapy, 6:973-985, 1999).

The virions are then harvested from the supernatant of transfected cells, isolated by freeze/thaw cycles and centrifugation. The virions may be purified by binding to a heparin-agarose column, eluted, and concentrated. For in vivo delivery, rAAV virions may be purified by fast performance liquid chromatography (FPLC).

Delivery of Virions to Target Cells

The rAAV virions formed from the tet-regulatable rAAV vectors may be delivered to target cells of the central or peripheral nervous system, or both, or any target cell from which the therapeutic protein can have an effect on a nervous system disorder. Preferably, the rAAV virions are added to the cells at the appropriate multiplicity of infection according to standard transduction methods appropriate for the particular target cells. Titers of rAAV virions to administer can vary, depending upon the target cell type and the particular viral vector, and may be determined by those of skill in the art without undue experimentation. rAAV virions are preferably administered to the cell in a therapeutically-effective amount. rAAV virions may be administered in a physiologically acceptable carrier. In general, a "physiologically acceptable carrier" is one that is not toxic or unduly detrimental to cells. Exemplary physiologically acceptable carriers include sterile, pyrogen-free, phosphate buffered saline. Physiologically-acceptable carriers include pharmaceutically-acceptable carriers.

The rAAV virions may be delivered to a target cell by any method known to one of skill in the art, including, but not limited to injection into the delivery site tissue. By way of example, for delivery to a specific region of the central nervous system, the rAAV virions may be administered by microinjection, infusion, convection enhanced delivery (CED), electroporation or other means suitable to directly deliver the composition directly into the delivery site tissue through a surgical incision. The delivery is accomplished slowly, such as at a rate of about 0.2-1 .mu.l per minute. Pursuant to the invention, administration of rAAV virions into selected regions of a subject's brain may be made by drilling a hole and piercing the dura to permit the needle of a microsyringe or micropipette to be inserted. A stereotaxic apparatus may be used to assist in delivering the virions to the specific target cells. Alternatively, rAAV virions may be delivered by lumbar puncture, for example, to the cerebral spinal fluid or delivered intraventricularly. The rAAV virions can be injected intrathecally into a spinal cord region. In another example, virions may be delivered to muscle in order to deliver rAAV to the terminals of motor neurons or sensory neurons.
 

Claim 1 of 13 Claims

1. A regulatable, recombinant adeno-associated viral vector comprising: SEQ ID NO:20 operably linked to a first nucleic acid encoding a protein that provides a therapeutic effect on a nervous system disorder; a second nucleic acid encoding a tetracycline-controlled transactivator; a tetracycline-off, regulatable promoter system comprising: a first promoter operably linked to the first nucleic acid; and a second promoter operably linked to the second nucleic acid; and inverted terminal repeats of adeno-associated virus, the inverted terminal repeats flanking the Promoter system; wherein the first and second promoters drive expression of the first and second nucleic acids in opposite directions, towards each other and away from the inverted terminal repeats in the vector.

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