EP1833975A2 - Regulated expression of transgenes in the central nervous system of mammals - Google Patents

Abstract

Recombinant adeno-associated virus (rAAV) vectors are provided for delivery of regulatable transgenes to the central nervous system (CNS) of a mammal. Also provided are methods of treatment of subjects with neurodegenerative disorders using the vectors, and kits for constructing or using the vectors or performing the methods of the invention. Transgene sequences are expressed from a promoter/enhancer region comprising one or more binding sites for a transcription factor that is responsive to a small molecule inducer. Both the transgene construct and a construct comprising the transcription factor are delivered to the target cells. The regulatable transgene may be delivered on the same rAAV vector as the transcription factor, or on a separate vector. The transcription factor may comprise two polypeptide chains, e.g. a DNA binding domain and a transcription activation domain, that form an active dimer in the presence of a dimerizer such as rapamycin or a non-immunogenic analog thereof. Vectors, methods and kits of the invention may be used to deliver genes such as AADC or GDNF to the brain of a subject with a neurodegenerative disorder, such as Parkinson's disease, where the expression of AADC or GDNF in the brain can subsequently be regulated by treatment of the subject with rapamycin or a rapamycin analog.

Description

REGULATED EXPRESSION OF TRANSGENES IN THE CENTRAL NERVOUS

SYSTEM OF MAMMALS

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Many human diseases are caused by the abnormal expression of genes. When 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. In addition, 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. In addition to supplementing under-expressed or defective endogenous genes, 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. 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 products themselves. Gene therapy is particularly preferred as a means for delivering

therapeutic proteins to the brain, since systemically administered proteins would be unlikely

to enter the brain due to the blood-bram-barrier, and direct stereotaxic injection into the brain

is impractical for repeated administration.

Adeno-Associated Virus-Mediated Gene Therapy

Viral vectors have been developed to assist in efficient delivery of transgenes to target

cells, a process referred to herein as transduction. One viral system that has been used for

gene delivery is adeno-associated virus (AAV). AAV is a parvovirus belonging to the genus

Dependovirus. AAV has several attractive features not found in other viruses. First, AAV

can infect a wide range of host cells, including non-dividing cells. The ability to infect non-

dividing cells makes AAV a particularly good choice for transduction of CNS tissue, e.g.

brain. Second, AAV can infect cells from different species. Third, AAV has not been

associated with any human or animal disease and does not appear to alter the biological

properties of the host cell upon integration. Indeed, it is estimated that 80-85% of the human

population has been exposed to the virus. Finally, AAV is stable at a wide range of physical

and chemical conditions, which facilitates production, storage and transportation.

The AAV genome is a linear, single-stranded DNA molecule approximately 4681

nucleotides (nt) in length. The AAV genome generally comprises an internal non-repeating genome flanked on each end by inverted terminal repeats (ITRs). The ITRs are

approximately 145 nt in length. The ITRs act as origins of DNA replication and as packaging

signals for the viral genome. The internal non-repeated portion of the genome includes two main open reading

frames, for the AAV replication (rep) and capsid (cap) genes. The rep and cap genes code for

proteins that allow the virus to replicate and package the viral genome into a virion. In

particular, 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. The

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) 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

genome persists as an episome or inserts into a host cell chromosome, but infectious virions

are not produced. Subsequent infection by a helper virus "rescues" the integrated genome,

allowing it to replicate and package its genome into infectious AAV virions. While AAV can

infect cells from different species, the helper virus must be of the same species as the host

cell. Thus, for example, human AAV will replicate in canine cells co-infected with a canine

adenovirus.

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) and inserting a heterologous

gene (a "transgene") between the ITRs. The transgene may be linked to a heterologous

promoter. Termination signals, such as polyadenylation sites, can also be included.

To produce a stock of infectious recombinant AAV (rAAV) containing the transgene,

a suitable producer cell line is transfected with an AAV expression vector containing a

transgene sandwiched between AAV ITRs. AAV helper functions and accessory functions

are also expressed in the producer cell. Helper functions are those functions normally

provided by AAV-encoded genes (e.g. rep and cap), and accessory functions are those functions normally provided by helper- virus when wild-type AAV (wtAAV) replicates in

cells co-infected with helper virus (e.g. adenovirus). The genes encoding helper functions in

wtAAV must be supplied separately because they are removed in construction of rAAV

vectors to make room for transgene sequences. In the presence of helper and accessory

functions the vector construct comprising the transgene and AAV ITRs is replicated and

packaged to form a recombinant AAV virion (rAAV). Production of rAAV stocks is further

described at U.S. Pat. Nos. 5,622,856; 6,001,650; 6,027,931; 6,365,403; 6,376,237;

5,945,335; 6,004,797; and 6,482,633, the disclosures of which are hereby incorporated by

reference in their entireties.

The rAAV stock so prepared can then be used to introduce the transgene into target

cells in vitro, or into a subject in vivo, by infecting the target cells with rAAV in the absence

of helper virus. Because the subject's cells lack the rep and cap genes and the accessory

function genes, rAAV vectors are replication defective in the target cell; that is, they cannot

further replicate and package their genomes. Similarly, without a source of rep and cap

genes, wtAAV cannot be formed in the subject's cells.

Regulation of Transgene Expression

One potential disadvantage to AAV-mediated gene therapy, and gene therapy in

general, is the inability to reverse the treatment, or modulate its effects, once it is

administered. Unlike delivery of traditional medications, which can be discontinued at any

time as necessary, successful gene therapy provides persistent long-term gene expression after

vector administration. The ability to shut down transgene expression is an important safety

consideration in treatment of human subjects. Furthermore, the ability to down-regulate transgene expression would be useful in ensuring the proper pharmacologic control (dosing)

of the transgene product in the subject.

Various systems for modulating transgene expression using small molecule inducers

have been developed. Rivera et al. (1996) Nat. Med. 2:1028-32; No et al. (1996) Proc. Natl.

Acad. ScL USA, 93: 3346-51; Gossen and Bujard (1992) Proc. Natl. Acad. ScI USA 89: 5547- 51; the GeneSwitch^® system (Valentis, Inc., Burlingame, California). These systems are

based on the use of engineered transcription factors whose activity is controlled by a small

molecule drug, and a transgene whose expression is driven by the regulated transcription

factor. One such system, based on induction by rapamycin (referred to herein as the

"dimerizer system"), involves formation of a functional transcription factor from two

synthetic fusion proteins dependent upon addition of rapamycin. Rivera et al. (1996) Nat.

Med. 2:1028-32. Rapamycin is an orally bioavailable small-molecule drug, closely related to

the natural-product immunosuppressant FK506, that binds with high affinity (200 pM) to the

cellular protein FKBP12, which complex then binds to FRAP. Although rapamycin has

intrinsic immunosuppressant activity, non-immunosuppressive analogs have been developed

that can be used with modified FRAP gene sequence to promote transcription in the dimerizer

system without undesirable immunosuppression. Pollock et al. (2000) Proc. Natl. Acad. ScL

USA 91 ': 13221-26.

The dimerizer-based system for in vivo genetic regulation is described in more detail

at Rivera et al. (1996) Nat. Med. 2:1028-32 and at Pollock et al. (2000) Proc. Natl. Acad. Sd.

USA 97: 13221-26. The dimerizer system has been adapted for use with viral vectors to

deliver genes to muscle (Ye et al. (1999) Science 283:88-91; Rivera et al. (1999) Proc. Natl.

Acad. ScL USA 96:8657-62; Johnston et al. (2003) MoI. Thar. 7:493-7), liver (Auricchio et al.

(2002) Gene Ther. 9:963-71), and eye (Auricchio et al. (2002) MoI. Ther. 6:238-42). The dimerizer system is also a component of the ARGENT Transcription Technology platform of

ARIAD Pharmaceuticals, Inc. (Cambridge, Massachusetts). This technology is further

disclosed at U.S. Pat. No. 6,043,082 to Crabtree et al., describing the ARGENT dimerizer-

based system generally, and U.S. Pat. No. 6,649,595 to Clackson et al., describing dimerizer-

based systems using rapamycin or a rapamycin analog, including composite DNA binding

domains, the p65 transcription activation domain and rapalogs with reduced

immunosuppression. The disclosures of U.S. Pat. Nos. 6,043,082 and 6,649,595 are hereby

incorporated by reference in their entireties.

The dimerizer system has the advantage of being fully humanized, in that the

sequences of the functional components of the transcription activation fusion proteins are all

derived from human proteins, thereby reducing the likelihood of adverse immune response in

humans. Rivera et al. (1999) Proc. Natl. Acad. Sd. USA 96:8657-62.

Parkinson's disease

Parkinson's disease (PD) is an example of a disease that may be amenable to gene

therapy, particularly AAV gene therapy. PD is the second most common neurodegenerative

disease in the United States, affecting over one million people. PD is characterized by a

decrease in spontaneous movements, gait difficulty, postural instability, rigidity and tremor.

These clinical signs are a direct result of the degeneration of the pigmented neurons (i.e.,

dopaminergic neurons) in the substantia nigra of the basal ganglia region of the brain. The

progressive degeneration of the substantia nigra leads to decreased availability of dopamine, as the pigmented neurons of the substantia nigra are the sites of synthesis of this important

catecholamine neurotransmitter. Dopamine is synthesized in the terminal nerve endings of the dopaminergic neurons

of the substantia nigra, specifically the substantia nigra pars compacta. The dopaminergic

nerves project into the corpus striatum, specifically innervating the putamen and the caudate

nucleus. Three enzymes are necessary for the efficient biosynthesis of dopamine: tyrosine

hydroxylase (TH), guanosine triphosphate cyclohydrolase I (GCH), and aromatic L-amino

acid decarboxylase (AADC). Tyrosine hydroxylase adds a hydroxyl group to the amino acid

tyrosine creating L-dihydroxyphenylalanine (L-dopa). GCH catalyzes the first and rate-

limiting step of the biosynthesis of BH₄, which is a necessary cofactor for TH activity. Lastly,

AADC removes the terminal carboxyl group of L-dopa to produce dopamine.

Treatment of PD currently involves oral administration of L-dopa, often in

combination with a peripheral inhibitor of AADC. As PD progresses, a majority of patients

experience a reduction in AADC content in affected regions of the brain (i.e., the substantia

nigra). Since AADC is required for conversion of L-dopa to dopamine, escalating doses of L-

dopa are required for therapeutic efficacy, but this often results in increased side effects.

Moreover, as the substantia nigra progressively deteriorates, AADC depletion continues

unabated, often reaching a level where therapeutic benefit derived from administration of L-

dopa is no longer realized.

AADC

One gene therapy-based approach to treatment of PD is to supply genes encoding one

or more enzymes involved in dopamine biosynthesis, such as AADC. The supplemental

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

hereby incorporated by reference in its entirety.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic

neurons in the substantia nigra and a severe decrease of dopamine in the striatum.

Hornykiewicz (1975) Natl. Inst. Drug Abuse Res. Monogr. Ser. 13-21. The 6-OHDA model

is produced by chemically lesioning the medial forebrain bundle that projects from the

substantia nigra to the striatum and histopathologically resembles PD. Ungerstedt (1971)

Acta Physiol. Scand. Suppl. 367: 69-93. Rats that are unilaterally 6-OHDA lesioned on one

hemisphere produce a rapid contralateral turning activity in response to therapeutic doses of

L-dopa or dopamine receptor agonists. Ungerstedt (1971). Previous studies have shown that

transferring the gene encoding human AADC (hAADC) to rat or non-human primate striatum and delivering L-dopa exogenously can restore dopamine to effective levels and lower L-dopa

requirements in animal models of Parkinson's Disease (PD). Bankiewicz et al. (2000) Exp.

Neurol. 164:2-14; Sanchez-Pernaute et al. (2001) MoI. Ther. 4:324-30.

GDNF

Another gene therapy-based approach to treatment of PD is to deliver genes that block

or slowdown the ongoing degenerative process. Glial cell line-derived neurotrophic factor

(GDNF) is a potent neurotrophic factor that has been shown to enhance DA neuron survival

both in vitro and in animal models of PD in vivo (Bjorklund et al., Brain Res. (2000) 886:82-

98; Bohn, M. C₅ Mo/. Ther. (2000) 1:494-496; et al., J. Neural Transm. Suppl. (2000)

58:181-191), the disclosures of which are hereby incorporated by reference in their entireties.

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.

2003/0050273, the disclosure of which is hereby incorporated by reference in its entirety.

Although expression of AADC or GDNF in the brains of Parkinson's patients maybe

beneficial, over-expression of the genes may lead to dangerous side effects. Typical vectors

used in gene therapy are designed to incorporate strong constitutive promoters that are

intended to maximize overall transgene expression in the subject to ensure a therapeutic

effect. Many previous studies have attempted to maximize expression from those few cells

that are transduced, which may be a reasonable goal when it is expected that transduction

efficiency will be relatively low, and when the transgene product is intended to be secreted

from the transduced cell into the general circulation. Several prior studies of dimerizer-

regulated transgene expression have involved such secreted proteins. Rivera et al. (1996)

Nat. Med. 2:1028-32; Rivera et al. (1999) Proc. Natl. Acad. Sd. USA 96:8657-62; Ye et al.

(1999) Science 283:88-91; Pollock et al. (2000) Proc. Natl. Acad. Sd. USA 97:13221-26;

Auricchio et al. (2002) Gene Ther. 9:963-71; Johnston et al. (2003) MoI. Ther. 7:493-7;

Auricchio et al. (2002) MoI. Ther. 6:238-42.

In contrast to secreted proteins whose production is pooled in the circulatory system,

therapeutic benefit may not be achieved if non-secreted transgene products are overexpressed in some cells but entirely lacking in others. Gene therapy using non-secreted proteins may

require that transgene expression be regulated to desirable levels within each transfected cell,

making it particularly important that the expression of such proteins be regulatable after

transduction.

Gene therapy of neurodegenerative diseases and other CNS disorders is an expanding

field (Tinsley and Eriksson (2004) Acta Neurol. Scand. 109: 1-8) and transgene regulation is likely to be a necessity for purposes of both dosing control and safety. Even though regulated

gene expression may not be necessary in the case of AADC therapy because the production of

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

termination of therapy if required. Regulation will likely be a requirement for gene delivery

of neurotrophic factors (such as GDNF) where over-expression could have deleterious effects,

with the required degree of control being determined by the specific application.

The need exists for a method of regulating expression of genes introduced into the

CNS (e.g. the brain) of a subject by gene therapy, and vectors enabling such regulation.

Specifically, the need exists for methods of regulating expression of transgenes in the brain of

a subject with a neurodegenerative disease, such as PD. Preferably, the regulatory system

would have a low background level of transgene expression under repressive conditions, but also exhibit a high induction ratio. The optimal system would also comprise functional

components derived exclusively from human proteins to minimize the chances of adverse

immune reactions during human gene therapy.

SUMMARY OF THE INVENTION

These and other needs in the art are met by the present invention, which 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.

In one aspect, the invention relates to recombinant AAV (rAAV) vectors in which

transgene expression can be regulated after the transgene has been transduced into the CNS

(e.g. the brain) of a subject. In one embodiment, regulation is effected using a transcription factor comprising two

polypeptide components, said transcription factor only being active when the two components

are bound to each other, and wherein the components bind to each only in the presence of an

inducer. In one embodiment, the two polypeptides comprise a DNA binding domain fusion

and an activation domain fusion. An exemplary DNA binding domain fusion comprises two DNA binding domains from the human transcription factor Zif268, a homeodomain derived

from Oct-1, and three drug-binding domains from the cytosolic receptor for FK506. An

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.

In one embodiment, a first rAAV vector comprises the transgene and a second rAAV

vector comprises the sequence of the transcription factor components. In another

embodiment a single rAAV vector comprises the sequences of the transcription factor

components and the transgene.

In one embodiment, regulation is accomplished by administration of an inducer, hi

another embodiment the inducer is a dimerizer, such as rapamycin or a non-

immunosuppressive analog thereof, e.g. AP21967.

Ia another aspect, the invention relates to methods of treatment of subjects with rAAV

vectors in which transgene expression can be regulated after the transgene has been

transduced into the CNS (e.g. the brain) of a subject. In one embodiment, the method of

treatment involves administration of an inducer, for example a dimerizer, such as rapamycin

or a non-immunosuppressive analog thereof, e.g. AP21967.

In yet another aspect, the invention relates to kits for constructing the vectors, or

performing the methods, of the invention. In one embodiment, the kit comprises a first rAAV

vector having a polylinker region for cloning a transgene of interest to a user. In another embodiment, 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. In yet

another embodiment, the kit comprises an inducer, for example a dimerizer such as

rapamycin or a non-immunosuppressive analog thereof, e.g. AP21967.

In one embodiment, the invention involves treatment of a human neurodegenerative

disease, e.g. Parkinson's disease (PD).

In some embodiments, the regulated transgene is AADC or GDNF, although in

general the regulated transgene can be any gene whose expression in the target tissue is

desired.

These and other embodiments of the subject invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.

DESCRIPTION OF THE DRAWINGS

FIG. IA is a diagram of a rAAV vector encoding the activation and DNA binding

domains of a transcription factor for regulation of transgene expression (AAV-CMV-TF).

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), expression of which can be regulated by addition of rapamycin or derivatives

thereof. 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

various rAAV constructs and subsequently treated with the rapamycin analog AP21967 at 0,

5 or 25 nM. A non-transduced control is also shown. "OD" values refer to the relative AADC expression (as measured by OD₄O₅ in an AADC expression ELISA) calculated using a

standard curve obtained with cells transduced with reference lots of AAV-CMV-hAADC2.

FIG. 3 is a timeline for experiments in which the rotational response to L-dopa is

measured in 6-OHDA lesioned rats as a function of rapamycin treatment.

FIG. 4 shows the rotational response to 5 mg/kg L-dopa in 6-OHDA lesioned rats,

expressed in contralateral turns per hour, as a function of time along the timeline shown at

FIG. 3. Rats are infused with either excipient alone (control) or with vectors encoding regulatable hAADC and the corresponding transcription factors. Data are not presented for

the first and second week. Infusion took place at day 0, as illustrated in FIG. 3, which is the

date from which the number of weeks is measured (e.g. "3 wks" refers to the day 21 day after

infusion). For simplicity, the groups of four daily rapamycin ("rap") injections shown in

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

a rapamycin-treated rat 7 weeks post-infusion with AAV vectors carrying the sequences

necessary for rapamycin-regulated expression of hAADC. The scale bar represents 75 μm.

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

brain sections from representative rats in three treatment groups obtained at 7 weeks post-

infusion: A: vector-infused (+) rap; B: vector-infused (-) rap; C: excipient-infused (+) rap.

As in FIG. 4, rats were infused with either excipient alone (FIG. 6C), or with vectors

encoding regulatable hAADC and the corresponding transcription factors (FIGS. 6 A and 6B).

Animals in FIGS. 6A and 6C were subsequently treated with rapamycin (as shown in FIG. 3)

but the animal in FIG. 6B was not. The left hemisphere is the site of both 6-OHDA lesion and intrastriatal vector (or excipient) infusions, and the right hemisphere shows endogenous

staining from intact rat AADC-positive fibers.

FIG. 7 shows the expression of AADC at 7 weeks post-infusion, as measured by

western blot analysis, in the three treatment groups discussed with reference to FIG. 6. The

top panels are images of the AADC and β-actin bands observed in gel electrophoresis of

proteins from the brains of representative animals from each of the three treatment groups, β-

actin is included as a control. The bar graph shows the average integrated image intensities,

and standard deviations, of the protein bands for the AADC bands for three animals in each

treatment group.

FIG. 8A is a diagram of an rAAV vector (AAV-TF-Z8-hGDNF) comprising a

transcript encoding the activation domain fusion and the DNA binding domain fusion of the

transcription factor, and a regulatable transcript encoding the transgene hGDNF, in which

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

expression of the DNA binding domain of the transcription factor is driven by an SV40

promoter on a separate transcript than expression of the activation domain, and the activation

domain is expressed from the opposite strand (i.e. in the opposite direction).

FIG. 8C is a diagram of a recombinant AAV vector encoding hGDNF, expression of

which is driven by the constitutive CMV promoter (AAV-CMV-hGDNF).

FIG. 9 shows GDNF expression, in picograms (pg), for HeLa D7-4 cells transiently

transfected with either a regulated TF-GDNF plasmid (AAV-TF-Z8-hGDNF) or a

constitutive CMV-GDNF plasmid (AAV-CMV-hGDNF), as a function of treatment with 0, 5

or 25 nM rapamycin. DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated,

conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and

pharmacology, within the skill of the art. Such techniques are explained fully in the literature.

See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and

Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition);

Sambrook, et al., Molecular Cloning: A Laboratory Manual (2 nd Edition, 1989); Methods In

Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's

Pharmaceutical Sciences, 18 th Edition (Easton, Pa.: Mack Publishing Company, 1990).

I. DEFINITIONS

AU scientific and technical terms used in this application have meanings commonly

used in the art unless otherwise specified. As used in this application, the following words or

phrases have the meanings specified.

It must be noted that, as used in this specification and the appended claims, the

singular forms "a", "an" and "the" include plural references unless the content clearly dictates

otherwise. Thus, for example, reference to "a vector" includes a mixture of two or more such vectors, and the like.

The term "transgene," as used herein, refers to any gene to be delivered to a target cell

regardless of the origin of the transgene, including, in some embodiments, additional copies

of, or sequence variants of, endogenous genes already present in the target cell. Transgenes

can be gene sequences or partial gene sequences obtained from any organism, genetically

engineered sequence variants of such genes, or synthetic (non-naturally occurring) DNA

sequences. 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.

A "regulatable transgene," as used herein, is a gene whose expression can be altered

by administration of an inducer after that gene is transduced into a target cell. Regulation of

therapeutic transgene expression provides pharmacologic control of the level of transgene

product.

"Expression cassette" refers to an assembly which is capable of directing the

expression of the sequence(s) or gene(s) of interest. The expression cassette includes a

promoter or promoter/enhancer which is operably linked to (so as to direct transcription of)

the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well.

Within certain embodiments of the invention, the expression cassette described herein maybe

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

of its origin or manipulation, is not associated with all or a portion of the polynucleotide with

which it is associated in nature. The term "recombinant" as used with respect to a protein or

polypeptide means a polypeptide produced by expression of a recombinant polynucleotide, hi

general, the gene of interest is cloned and then expressed in transformed organisms, as

described further below. The host organism expresses the foreign gene to produce the protein

under expression conditions.

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

mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory

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)

terminus. A coding sequence can include, but is not limited to, cDNA from viral, procaryotic

or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even

synthetic DNA sequences. A transcription termination sequence may be located 3' to the

coding sequence.

Typical "control elements," include, but are not limited to, transcription promoters,

transcription enhancer elements, transcription termination signals, polyadenylation sequences

(located 3' to the translation stop codon), sequences for optimization of initiation of

translation (located 5' to the coding sequence), and translation termination sequences.

The term "nucleic acid" includes DNA and RNA, and also their analogues, such as

those containing modified backbones (e.g. phosphorothioates, etc.), and also peptide nucleic

acids (PNA), etc. The invention includes nucleic acids comprising sequences complementary

to those described above (e.g. for antisense or probing purposes).

The term "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.

A number of transfection techniques are generally known in the art. See, e.g., Graham et al.

(1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual,

Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular

Biology, Elsevier, and Chu et al. Gene 13:197, 1981. Such techniques can be used to

introduce one or more exogenous DNA moieties, such as a plasmid vector and other nucleic

acid molecules, into suitable host cells. The term refers to both stable and transient uptake of

the genetic material.

The term "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. The term "heterologous" as it relates to nucleic acid sequences such as gene sequences

and control sequences, denotes sequences that are not normally joined together, and/or are not

normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid

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. For example, a

heterologous region of a nucleic acid construct could include a coding sequence flanked by

sequences not found in association with the coding sequence in nature. Another example of a

heterologous coding sequence is a construct where the coding sequence itself is not found in

nature (e.g., synthetic sequences having codons different from the native gene). Similarly, a

cell transformed with a construct which is not normally present in the cell would be

considered heterologous for purposes of this invention. Allelic variation or naturally

occurring mutational events do not give rise to heterologous DNA, as used herein.

The term "control elements" refers collectively to promoter regions, polyadenylation

signals, transcription termination sequences, upstream regulatory domains, origins of

replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively

provide for the replication, transcription and translation of a coding sequence in a recipient

cell. Not all of these control elements need always be present so long as the selected coding

sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

The term "promoter region" is used herein in its ordinary sense to refer to a nucleotide

region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived

from a gene which is capable of binding RNA polymerase and initiating transcription of a

downstream (3 '-direction) coding sequence.

"Operably linked" refers to an arrangement of elements wherein the components so

described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding

sequence. The control elements need not be contiguous with the coding sequence, so long as

they function to direct the expression thereof. Thus, for example, intervening untranslated yet

transcribed sequences can be present between a promoter sequence and the coding sequence

and the promoter sequence can still be considered "operably linked" to the coding sequence.

By "isolated" when referring to a nucleotide sequence, is meant that the indicated

molecule is present in the substantial absence of other biological macromolecules of the same

type. Thus, an "isolated nucleic acid molecule which encodes a particular polypeptide" refers

to a 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

or moieties which do not deleteriously affect the basic characteristics of the composition.

A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral

vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct,"

"expression vector, " and "gene transfer vector," mean 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. Thus, the term includes cloning and expression vehicles, as well as

viral vectors.

By "subject" is meant any member of the subphylum chordata, including, without

limitation, humans and other primates, including non-human primates such as chimpanzees

and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and

horses; domestic mammals such as dogs and cats; laboratory animals including rodents such

as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as

chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not

denote a particular age. Thus, both adult and newborn individuals are intended to be covered. By "therapeutically effective dose or amount" of an AAV vector encoding a

regulatable transcription factor and/or transgene is intended an amount that when the AAV

vector is administered as described herein, brings about a positive therapeutic response, for

example, ameliorates symptoms or prevents progression of a neurological disorder. For

example, administration of a therapeutically effective amount of an AAV vector, which

results in the expression of a therapeutic agent (e.g., AADC, GDNF) in a subject being treated

for Parkinson's disease, may ameliorate symptoms, e.g., improve motor function or reduce

resting tremor. The exact amount of the AAV vector required will vary from subject to

subject, depending on the species, age, and general condition of the subject, the severity of the

condition being treated, and the particular composition used, mode of administration, and the

like.

II. MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to be understood that this

invention is not limited to particularly exemplified molecules or process parameters as such

may, of course, vary. It is also to be understood that the terminology used herein is for the

purpose of describing particular embodiments of the invention only, and is not intended to be

limiting. In addition, the practice of the present invention will employ, unless otherwise

indicated, conventional methods of virology, microbiology, molecular biology, recombinant

DNA techniques and immunology all of which are within the ordinary skill of the art. Such

techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular

Cloning: A Laboratory Manual (2nd Edition, 1989); DNA Cloning: A Practical Approach,

vol. I & π (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); A Practical Guide to Molecular Cloning (1984); and Fundamental Virology, 2nd Edition, vol. I & II (B.N. Fields

and D.M. Knipe, eds.). Although a number of methods and materials similar or equivalent to

those described herein can be used in the practice of the present invention, the preferred

materials and methods are described herein.

The present invention provides vector constructs, methods and kits for regulation of

transgene expression in a target cell. In one embodiment, the sequences necessary for

transduction of a regulatable transgene in a target cell are provided on a plurality of separate

recombinant AAV vector molecules. 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

transgene in a target cell. Example 3 demonstrates the construction and use of one such

single- vector embodiment of the present invention. hi one embodiment, 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

embodiment, a regulatable form of the GDNF gene is delivered to target cells. Example 3

demonstrates delivery of a regulatable form of the GDNF gene.

The term transgene, as used herein, refers to any gene to be delivered to a target cell

regardless of the origin of the transgene, including, in some embodiments, additional copies

of, or sequence variants of, endogenous genes already present in the target cell. Transgenes

can be gene sequences or partial gene sequences obtained from any organism, genetically

engineered sequence variants of such genes, or synthetic (non-naturally occurring) DNA sequences. 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. A regulatable transgene, as used herein, is a gene whose expression can be altered by

administration of an inducer after that gene is transduced into a target cell. Regulation of

therapeutic transgene expression provides pharmacologic control of the level of transgene

product. In preferred embodiments the inducer is a small molecule drug. One such small

molecule drug is rapamycin.

In one embodiment, the regulatable gene expression system of the present invention

makes use of the specific dimerization of the immunophilin FKBP and the lipid kinase

homolog FRAP in the presence of rapamycin or AP21967, a non-immunosuppressive

rapamycin analog, to produce an active transcription factor complex that specifically activates

an inducible promoter. Rivera et al. (1996) Nat. Med., 2:1028-32. The structure of AP21967

is provided below.

AP21967

AP21967 (molecular mass 1017.4 Da) is the 7-methylindolyl analog of Compound 69

in U.S. Pat. No. 6,649,595 (the '595 patent), and can be prepared as described in Example 5

therein by substituting 7-methylindole for dimethoxybenzene. AP21967 is identical to Compound 71 of the '595 patent except that AP21967 has a 7-methyl group on the indole ring.

Other rapamycin analogs can also be used without deviating from the scope of the

present invention. Rapamycin analogs that do not interact with the endogenous FRAP in the

subject will not have the potentially undesirable immunosuppressive and cell-cycle inhibitory

effects of rapamycin. Pollock et al. (2000) Proc. Natl. Acad. ScL USA 97:13221-26. Mutant

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).

Regulated Expression of AADC

Experiments are performed using AAV vector-mediated, rapamycin-dependent

regulation of expression of an AADC transgene in HeLa D7-4 cells in vitro, and in a rodent

model of Parkinson's disease (PD) in vivo. Expression of the human AADC (hAADC)

transgene is made dependent on reconstitution of a functional transcription factor (TF) by the

dimerizer rapamycin using vectors described further in Example 1 and FIGS. IA and IB. A

transcription factor AAV vector is used to deliver the DNA binding domain and the activation

domain of the transcription factor, and an expression AAV vector is used to deliver the

hAADC transgene under the control of a regulatable promoter.

AADC Regulation in vitro

Experiments demonstrating dimerizer-dependent AADC expression in human cells in

vitro are described in Example 1. The dimerizer used in Example 1, AP21967, is a non-

immunosuppressive rapamycin analog that is approximately 3-fold less potent than rapamycin. Results are shown at FIG. 2, in which the right-most data show rapamycin-dose-

dependent hAADC expression (from AAV-Z 12-hAADC) in cells co-transduced with a vector

encoding the dimerizer-dependent transcription factor (AAV-CMV-TF). The ELISA data

show that AADC expression is 9-fold higher in the presence of 25 nM AP21967 than in the

absence of dimerizer. 25 nM is the maximally effective concentration of AP21967, giving

plateau levels of transgene expression in vitro. hAADC expression from the regulatable

promoter at 25 nM AP21967 is roughly half the level from hAADC under control of the

constitutive CMV promoter (AAV-CMV-hAADC2) (FIG. 1C, and described at Sanftner et al.

(2004) MoI. Ther. 9:403-9).

Cells harboring both vectors (AAV-Z 12-hAADC and AAV-CMV-TF) exhibit only

low level AADC production in the absence of AP21967, i.e. the system is not "leaky."

Absence of leakiness can be critical in gene therapy to ensure that transgenes can be totally

shut off if necessary, and to provide a wide range of pharmacologic regulation.

AADC Regulation in vivo

The same vectors used to demonstrate AAV-mediated transduction of dimerizer-

regulated hAADC in vitro (FIGS. IA and IB) are tested in vivo in the 6-hydroxydopamine (6-

OHDA) rat model of Parkinson's disease, a surgical model of striatal denervation, as

described in Example 2. The treatment schedule is presented schematically at FIG. 3.

Parkinsonian rats are transduced with both AAV-CMV-TF and AAV-Z 12-hAADC and their

rotational behavior is then measured after a series of treatments with rapamycin, as shown in

FIG. 4.

Rapamycin is used in the in vivo experiments, rather than AP21967 as was used in

vitro, simply because the proper dosage of rapamycin had been previously determined. The dimerizer rapamycin has many favorable properties for use as an inducer in human gene

therapy, but its inherent immunosuppressive properties may limit its usefulness. Non-

immunosuppressive analogs of rapamycin, such as AP21967, may be superior inducers of

transgene expression, particularly in treatment of human subjects. The proper dosage of

AP21967 in animals and human subjects in vivo may be determined by standard experimental

methods, including clinical trials.

Transduction of the regulated hAADC vector in combination with administration of

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

an altered rotational response to 5 mg/kg L-dopa that is reversible upon rapamycin

withdrawal in the vector-infused (+) rap group. The rotational response of the vector-infused (+) rapamycin group to L-dopa is significantly increased above both the vector-infused (-)

rapamycin group and the excipient-infused control group at weeks 3, 5, and 7 (P < 0.001)

immediately following rapamycin treatment, but returns to near control levels at weeks 4 and

6. These data demonstrate that transgenic hAADC expression can be reversibly regulated in

vivo in a mammalian brain to affect behavioral phenotype.

Real-time quantitative PCR confirms that the rats in the vector-infused (+) rapamycin

group and the vector-infused (-) rapamycin group are transduced with equal numbers of

copies of the hAADC gene, eliminating differential transduction efficiency as a possible

explanation for the different results observed with the two groups.

Immunohistochemistry and protein expression assays performed at the endpoint of the

study (Table 1 and FIGS. 5, 6 and 7) confirm the behavioral results, as discussed below in

detail. FIG. 5A demonstrates strong immunohistochemical staining for AADC, and thus

efficient transgene expression, in medium spiny neurons in the striatum of a rat from the

vector-infused (+) rapamycin group, whereas FIG. 5B shows only very low level AADC

expression in a rat from the vector-infused (-) rapamycin group.

FIGS. 6A-6C present low resolution images of whole mounted brain sections

immunohistochemically stained for AADC. The results give simple visual confirmation of

significantly higher level expression of AADC in the left (treated) hemisphere of a rat from the vector-infused (+) rapamycin group (FIG. 6A) compared with the left (treated)

hemisphere of a rat from the vector-infused (-) rapamycin group (FIG. 6B). The results for

the animal from the vector-infused (-) rapamycin group is similar to the results for the control

excipient-infused animal (FIG. 6C).

Immunohistochemical stereological analysis confirms that expression levels measured

by positive cell counts are significantly increased when vector is administered in combination

with rapamycin. Table 1 presents the results of transgene-derived immunostaining and

estimated positive cell counts by quantitative stereology for rapamycin-induced and

uninduced animals. Rapamycin-induced animals show approximately twice the anterior-to-

posterior spread of AADC immunostaining, volume of spread of AADC immunostaining, and

AADC positive cell number as compared to uninduced animals.

Total protein analysis shows an even greater disparity in protein levels. As shown at

FIG. 7, Western blot analysis of hAADC enzyme levels after gel electrophoresis of striatal

protein samples shows significantly higher hAADC expression in the (+) rapamycin group as

compared to the (-) rapamycin group. When endogenous AADC expression is subtracted

from the data plotted in FIG. 7 the level of hAADC is 88% lower in the (-) rapamycin group

as compared to the (+) rapamycin group. The results of the experiments described in Example 2 indicate that hAADC gene

expression is induced by the dimerizer rapamycin, although the low level expression observed

in the uninduced animals suggests some "leakiness" in the regulatory system in vivo. The

reason for the system's leakiness is not clear, and without intending to be limited by theory it

may be caused by unregulated expression from the minimal IL-2 promoter in the absence of

bound transcription factor. However, the low level of hAADC observed in the absence of

induction is not enough to elicit a behavioral response to a sub-therapeutic dose of L-dopa,

suggesting that a small amount of gene expression produced by the inducible promoter in the

absence of inducing agent may be tolerable for this particular application.

The proper dosage of rapamycin (or analog) to effect the desired level of transgene

induction in vivo may be determined by experimentation on a case-by-case basis, as is

common with therapeutic protocols. Dosage may be adjusted by trial and error based on

phenotypic measurements or on surrogate markers. Dosages may also be adjusted to achieve

a predetermined target concentration of rapamycin in the target tissue or in the blood of the

subject. When introduced by intravenous injection, 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,

1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg. In the case of treatment of human subjects, dosage

may be determined by reference to the results of clinical trials or extrapolated from animal

data.

Recombinant AAV vectors can be surgically introduced at various locations in the

brain, depending on the transgene being delivered, its mechanism of action, and the desired

target cells. For example, the basal ganglia are groups of neurons positioned subcortically.

They include the caudate nucleus, putamen, and globus pallidus. The caudate nucleus and the

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

nigra pars compacta (SNpc) and the substantia nigra pars reticulata. The SNpc is the normal

site of dopamine biosynthesis; degeneration of the SNpc is a hallmark of PD. By transducing

neurons of the putamen or caudate nucleus of the corpus striatum with genes encoding

enzymes in the dopamine biosynthetic pathway, such as AADC, dopamine synthesis can be

restored, thereby overcoming a functionally diminished SNpc network.

Corpus striatal cells can be transduced using a variety of techniques known in the art.

For example, stereotaxic injection is a common surgical technique used by neurosurgeons to

administer various compounds to the CNS. Direct injection can also be employed; if using this technique, anatomical maps derived from CT, PET, or MRI scans can be used by the

surgeon to aid in selecting the site or sites of injection. Other techniques, including

convection-enhanced delivery (described in detail in U.S. Pat. No. 6,309,634, hereby incorporated by reference in its entirety), can be employed in the methods of the present

invention to deliver rAAV virions to the CNS.

The experiments described in Examples 1 and 2 involve a dual-vector embodiment of

the present invention, in which separate vectors are used to deliver the transcription factor

fusion proteins and the transgene. In other embodiments, different genes are used in place of

hAADC in the expression vector. A maximum of approximately 5000-5200 nucleotides (nt)

can be packaged in an AAV virion, with approximately 4500 nt being optimal. Dong et al.

(1996) Hum. Gene Ther. 7:2101-12. Given the other required sequence elements of rAAV

expression vectors like the one illustrated at FIG. IB (e.g. ITR regions, promoter,

transcription factor binding sequences, etc.), transgene sequences in the expression vector

may be no longer than approximately 2500 nucleotides (nt). Within this size constraint, any desired transgene can be delivered using the rAAV expression- vector constructs of a dual- vector embodiment of the present invention. Optimized expression vector variants with

shorter ITR, promoter or transcription factor binding regions may be constructed to enable

delivery of longer transgene sequences.

Results from Examples 1 and 2 demonstrate that expression of a dimerizer-dependent

hAADC transgene can be regulated in human cells in culture, and in rat neurons in vivo, using

a dual- vector rAAV-mediated embodiment of the present invention.

GDNF Regulation In a Single-Vector System

As described in Example 3, experiments are performed using a single-vector

embodiment of the present invention, in which a single rAAV vector is designed to express

all of the proteins necessary for dimerizer-dependent regulation of expression of a GDNF

transgene. A diagram of a regulated rAAV hGDNF expression vector is provided at FIG 8 A,

and 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

vector that is not used in Example 3.) Plasmid vectors are transiently transfected into HEK-

293 cells in vitro. Cells are then treated with media containing 0, 5 or 25 nM rapamycin.

Results of GDNF ELISAs performed three days after transfection are presented at

FIG. 9. The data show rapamycin-dose-responsive expression of GDNF in cells transfected

with the regulated construct ("TF-GDNF plasmid," which refers to pAAV-TF-Z8-hGDNF),

with a maximum observed expression approximately one fourth the level of expression from

the constitutively expressed GDNF ("CMV-GDNF plasmid," which refers to pAAV-CMV- hGDNF). GDNF expression from pCMV-GDNF is not increased by addition of rapamycin.

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. The

results suggest that expression of GDNF may also be regulatable in subjects in vivo using a

regulatable AAV-GDNF vector. The single vector of Example 3 fills the rolls of both

transcription factor vector and expression vector in the dual-vector embodiment discussed

above. Treatment with a single vector has the advantage of requiring only one transduction

event to introduce a regulatable GDNF, as compared to the two-vector approach, which

requires co-transfection of target cells with two vectors. This advantage of the single- vector

embodiment is particularly significant for protocols that give only a low transduction

efficiency, such as gene therapy methods where only a very small proportion of target cells

would be expected to be simultaneously transduced by both vectors. For example, assuming

independent transduction efficiencies, only 0.25% of cells would be transduced with both

vectors if the overall transduction efficiency was 5% for each vector individually.

Because a vector to be packaged in an AAV virion cannot be longer than

approximately 5000-5200 nucleotides (nt), single- vector regulatable constructs will only be

practical for delivery of relatively short transgenes. For example, with the particular choice of

transcription factor fusion proteins and regulatory elements used in the rAAV vector

illustrated at FIG. 8A (as used in Example 3), the transgene (GDNF) sequence is

approximately 600 nt long. It is possible that longer transgene sequences, e.g. up to 850 or

even 1000 nt, may be delivered using optimized single- vector regulatable AAV-constructs

with more efficient placement of sequence elements, and in which unnecessary nucleotides

are eliminated from fusion proteins and regulatory elements. Within the size constraints for

AAV-packaging, any desired transgene can be delivered using the single-vector constructs of the present invention. Transgenes may comprise active sub-fragments of desirable genes,

rather than full-length genes, to facilitate single- vector delivery. The dose of rAAV virions required to achieve a particular therapeutic effect, e.g., the

units of dose in vector genomes/per kilogram of body weight (vg/kg), may depend on several

factors including, but not limited to: the route of rAAV virion administration, the level of

transgene expression required to achieve a therapeutic effect, and the stability of the

heterologous gene product. One of skill in the art can determine a rAAV virion dose range to

treat a subject having a particular disease or disorder based on the aforementioned factors, as

well as other factors that are well known in the art. In some embodiments of the present

invention, the proper dose of rAAV used to effect transduction in a mammal may range from

1 XlO⁸ vg/kg to 1 XlO¹⁵ vg/kg, preferably 4 XlO⁹ vg/kg to 4 XlO¹² vg/kg, although higher or

lower does may be employed as determined by experimentation. Although AADC and

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. Other genes or portions of

genes that might be expected to provide a beneficial (e.g. therapeutic) effect may be

introduced into the brain using the vectors, methods and kits of the present invention,

provided that the sequence is short enough to fit into an AAV vector construct that can be

packaged into AAV virions.

As discussed above, AADC (OMBvI 107930, EC 4.1.1.28, Genbank Accession

No. M76180) is a transgene involved in dopamine biosynthesis. Other potential transgenes involved in dopamine biosynthesis are tyrosine hydroxylase (TH) (OMIM 191290, EC

1.14.16.2, Genbank Accession No. X05290) and guanosine triphosphate cyclohydrolase I

(GCH) (OMM 600225, EC 3.5.4.16, Genbank Accession No. NM_000161). Still other

potential transgenes 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,

Genbank Accession No. HSU78110), and persephin (OMIM 602921, Genbank Accession

No. AF040962.). Saarma et al. (1999) Microscopy Res. Tech. 45:292-302. Still other

transgenes include IL-IO (OMM 124092, Genbank Accession No. M57627).

OMIM numbers refer to the Online Mendelian Inheritance in Man database

maintained by Johns Hopkins University, available on the internet through the National

Library of Medicine website at www.ncbi.nlm.nih.gov/entrez. The contents of all OMM

entries cited herein, and sequences cited by accession numbers, are hereby incorporated by reference in their entireties.

Genbank accession numbers are provided for representative complete cDNA

sequences only and are not intended to limit the scope of the invention. In particular,

potential transgenes include other sequences reported for the gene in Genbank, full-length and

subfragments of the natural genes, homologous genes from other species, allelic variants of

these sequences, and either naturally-occurring or artificially created mutant forms of the

genes.

Transgenes also include genes for treatment of other neurodegenerative diseases, such

as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Canavan disease, cerebral ischemia, progressive supranuclear palsy, Lewy body

dementia, Shy-Drager syndrome, ADDS dementia syndrome, essential tremor, dystonia,

corticobasal degeneration, multiple system atrophy and retinal degeneration (e.g. macular

degeneration, diabetic retinopathy, retinitis pigmentosa, glaucoma).

The inducer is preferably selected from among compounds that can be delivered to a

human subject without significant toxicity or side effects. In preferred embodiments for

treatment of neurological or other CNS diseases, the inducer is able to cross the blood-brain barrier. In more preferred embodiments the inducer is a dimerizer, for example rapamycin or

a non-immunosuppressive analog thereof.

In some embodiments the inducer is delivered parenterally, e.g. by subcutaneous,

intramuscular, intraocular or intravenous injection. In preferred embodiments the inducer is

one that can be delivered relatively conveniently, such as orally, topically, by nasal delivery (nasal spray), by aerosol/pulmonary delivery (inhaler), by ocular delivery (eye drops) or by

any other convenient mode of delivery. Inducer may also be delivered continuously or semi-

continuously using a transdermal patch, a subcutaneous implant, an implantable osmotic

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

of several methods known in the art for AAV virion production. Wild-type AAV and helper viruses can be used to provide the necessary replicative functions for producing rAAV

virions, or a plasmid can be used to supply the either helper function genes (e.g. pHLP 19),

the accessory function genes (e.g. pladeno 5), or both in the case of the triple transduction

method. See, e.g., U.S. Pat. Nos. 5,139,941; 5,622,856; 6,001,650 and 6,004,797, the

disclosures of which are hereby incorporated by reference in their entireties.

For in vivo delivery, rAAV virions are formulated into pharmaceutical compositions

comprising an appropriate dose of one or more rAAV virions and a pharmaceutically

acceptable excipient. Such excipients include any pharmaceutical agent that does not itself

induce the production of antibodies harmful to the individual receiving the composition, and

which may be administered without undue toxicity. Pharmaceutically acceptable excipients

include, but are not limited to, liquids such as water, saline, glycerol and ethanol.

Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts

such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally,

auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the

like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable

excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub.

Co., NJ. 1991). hi some embodiments, rAAV virions of the present invention are supplied as

pharmaceutical compositions comprising excipients that enhance viral stability during

prolonged storage and exposure to repeated freeze-thaw cycles, and that reduce adherence of vector to the infusion device, hi preferred embodiments, excipients exhibit low toxicity in the

target tissue, e.g. the CNS. For example, virions can be stored and supplied in buffer

comprising pluronic F-68 (BASF Corp., Mount Olive, NJ) at 0.01% to 0.0001%, preferably 0.001%. Additional compositions and excipients are described in U.S. Pat. Nos. 6,759,050

and 6,764,845, the disclosures of which are hereby incorporated by reference in their

entireties. hi another aspect the present invention relates to kits for construction of AAV vectors

for regulated expression of transgenes. hi some embodiments, one or more vectors

substantially identical to those shown in FIGS. IA and IB, or FIGS. 8 A or 8B, are included in

such kits.

hi other embodiments, vectors similar to those shown in FIGS. IB, 8A or 8B are

included in such kits except that the AADC and GDNF coding sequences are replaced by

convenient cloning sequences, such as a polylinker. Expression vectors similar to that shown

in 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

vector, and then prepare rAAV virions for transduction.

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,

such as the plasmids pHLP 19 and pladeno 5, as described more fully in U.S. Pat.

Nos. 6,001,650 and 6,004,797.

Kits may also contain instructions for use.

hi some embodiments, 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

therapeutic effect. Generally speaking, "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

desired outcome or clinical endpoint, such that a subject's disease or disorder shows clinical

improvement, often reflected by the amelioration of a clinical sign or symptom relating to the

disease or disorder, hi the case of Parkinson's disease, a therapeutic effect can be an

improvement in motor function (e.g., fine motor tasking) manifested, for example, by

improvement in manual dexterity. Alternatively, a reduction in resting tremor can also be a

sign of amelioration of PD. There are several other art-recognized observable and measurable

endpoints to determine therapeutic efficacy (i.e., a therapeutic effect) for a particular

treatment of PD.

In human patients displaying clinical signs and symptoms of PD, clinicians often rely

upon the well-known Unified Parkinson's Disease Rating Scale (UPDRS) to assess the

severity of disease and also to measure the therapeutic efficacy of a particular treatment modality. Analogous to the UPDRS system, scientists assess PD features in primate models

of PD using the Primate Parkinsonism Rating Scale (PPRS, also known as Clinical Rating Score - CRS), which measures, among other features, fine motor tasking, resting tremor,

bradykinesia, hypokinesia, and muscular rigidity. The PPRS system is described in Langston

et al. (2000) Ann. Neurol. 47:S79-89.

III. EXPERIMENTAL

Below are examples of specific embodiments for carrying out the present invention.

The examples are offered for illustrative purposes only, and are not intended to limit the

scope of the present invention in any way. Efforts have been made to ensure accuracy with

respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and

deviation should, of course, be allowed for.

EXAMPLE 1

REGULATED EXPRESSION OF AADC IN VITRO

Regulation of an AADC transgene in vitro is achieved as follows.

AA V Vectors

Two vectors are constructed to achieve dimerizer-dependent expression of hAADC:

AAV-CMV-TF and AAV-Z12-hAADC. FIG. IA is a diagram of AAV-CMV-TF, in which

the cytomegalovirus (CMV) enhancer/promoter drives expression of a bicistronic message

encoding activation domain and DNA binding domain fusion proteins. The activation

domain fusion protein contains the rapamycin binding domain (FRB*) of human FRAP fused

to the transcriptional activation domain derived from the p65 subunit of NFKB (p65). The

particular FRB domain used in this embodiment (FRBT₂O9SL)₃ and illustrated in FIG. IA (FRB*), carries a T to L mutation at position 2098 when compared to the wild-type FRB

sequence. Pollock et al. (2000) Proc. Natl. Acad. ScL USA 97:13221-26. 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

virus. The DNA binding domain fusion protein contains two DNA binding domains from the

human transcription factor Zif268, a homeodomain derived from Oct-1 (ZFHDl), and three

drug binding domains from the cytosolic receptor for FK506 (3xFKBP). FIG. IB is a

diagram of AAV-Z 12-hAADC, in which expression of human AADC (hAADC) is controlled

by 12 binding sites for the transcription factor fused to a minimal IL-2 promoter. AA V2

inverted terminal repeat (ITR), hAADC coding sequence (hAADC) and human growth

hormone polyadenylation (pA) are indicated.

In vitro expression analysis

The ability to regulate hAADC gene expression is assayed in vitro by co-transducing

HeLa D7-4 cells with a 1:1 ratio AAV-CMV-TF and AAV-Z12-hAADC, and subsequently

treating the cells with the rapamycin analog AP21967 (ARIAD Pharmaceuticals, Inc., Cambridge, Massachusetts) at 0, 5 or 25 nM. Results are presented at FIG. 2, which includes

the results of various control experiments, including non-transduced cells, cells transduced

with transcription factor vector alone (AAV-CMV-TF), cells transduced with a constitutively

expressed hAADC vector (AAV-CMV-hAADC2), and cells transduced with the regulated

hAADC vector (AAV-Z12-hAADC) but lacking the transcription factor vector.

Experimental details regarding experimental methods used in Example 1 are as

follows. Vector Construction

AAV-CMV-TF has been described previously (AAV-CMV-TF INc, Auricchio et al.

(2002) MoI. Titer. 6:238-42). AAV-Z12-hAADC is created by replacing the CMV enhancer

and promoter of pAAV-hAADC (Sanftner et al. (2004) MoI. Ther. 9:403-9) with a region of

Z12-IL2-SEAP (Rivera et al. (1996) Nat. Med. 2:1028-32) containing 12 binding sites for the

ZFHDl DNA binding domain upstream of a minimal IL-2 promoter.

Recombinant vector production

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

centrifugation. Grimm et al. (2003) Blood 102:2412-9. Briefly, cell harvests containing

rAAV are microfluidized and filtered through 0.2-μm filters. Vector is purified from the

clarified cell lysates by CsCl density gradient centrifugation, concentrated by ultrafiltration,

and diafiltration into phosphate-buffered saline containing 5% sorbitol, pH 7.4 (PBS-

sorbitol), with 0.001% pluronic F-68 (BASF Corp., Mount Olive, NJ) added to prevent vector

loss in delivery catheters. Vector purity is assessed by SDS-PAGE. Purified rAAV vector

used in this study essentially shows only VPl, VP2, and VP3 by silver staining of SDS-

PAGE gels. Titer is determined by Q-PCR analysis of vector genomes.

In vitro AADC analysis by expression ELISA

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

rAAV vectors and treated with three different doses of AP21967 (0, 5, 25 nM). n = 3 for

each group. Cells are seeded in a 96-well plate 24 hr before transduction. Cells are transduced with an MOI of 10⁴ vg/cell (of each vector when multiple vectors are used) in 100

μl of complete medium.

ELISA is performed on transduced cells 48 h post-transduction. Briefly, media are

aspirated and cells are washed with PBS. Cells are fixed with 4% paraformaldehyde and

incubated for 20 min at room temperature. Cells are washed with PBS, while shaking, and

blocked with blocking buffer (3% goat serum, 0.5% Triton X-IOO in PBS), then incubated for

60 min at room temperature while shaking. 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

PBS) and incubated in a shaker for 60 min at room temperature. Plates are washed with wash

buffer and the secondary antibody (goat anti rabbit IgG-AP (Vector Labs, Burlingame,

California), 1 : 1000 in wash buffer) is incubated in a shaker for 30 min at room temperature.

Plates are washed with wash buffer. Substrate solution is added [IX p-nitrophenyl

phosphate,lX levamisole (quenching reagent), in substrate buffer (10OmM NaHCO₃, pH

10.0) (Vector Labs, Burlingame, California)] and the plate is incubated at room temperature

for 30-60 min, then read in a plate reader at OD₄₀₅. Data from this hAADC expression

ELISA are reported as the relative optical density measured in cells transduced with vector in

comparison to a dose response curve for transduction with reference lots of AAV-CMV-

hAADC2.

EXAMPLE 2

REGULATED EXPRESSION OF AADC /N VIVO

The same vectors used to demonstrate AAV-mediated transduction of dimerizer-

regulated hAADC in vitro (Example 1, 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

clearance. Gallant-Haidner et al. (2000) Titer. DrugMonit. 22: 31-5.

All rats used in this example are unilaterally 6-OHDA lesioned on the left side. There

are three experimental groups: an excipient-infused control group (excipient-infused (+)

rapamycin treatment), a vector-infused (-) rapamycin treatment control group, and a vector-

infused (+) rapamycin treatment group. 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.

A 1:1 mixture of AAV-CMV-TF and AAV-Z12-hAADC (3xlO¹⁰ viral genomes (vg)

of each) is infused ipsilaterally into unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats.

Excipient alone is infused in the excipient-infused control group. Induction of transgene

expression is achieved via intraperitoneal injection of rapamycin at designated time points, as

discussed below.

FIG. 3 shows the experimental timeline for the experiments described in this example.

Prior to rAAV treatment, a baseline rotational test upon administration of L-dopa (5 mg/kg)

is given to all groups. Vector or excipient is infused intrastriatally on day 0. On day 17 rats

are either induced with rapamycin or treated with diluent. Induction consists of four

consecutive days of IP injection of 10 mg/kg/day of rapamycin. (Rapamycin is administered

for 4 consecutive days to ensure maximal circulating levels in the brain.) Arrows in FIG. 4

indicate the beginning of the four day rapamycin induction. On day 21 rats are again tested

for a rotational response to 5 mg/kg L-dopa. Rats are allowed to recover from rapamycin for

1 week and then tested for a response on day 28. Rats are induced a second time on day 31

and tested for a rotational response on day 35. After recovering from rapamycin for a week, the rotational test is repeated on day 42. On day 45 rats receive the third and final course of

induction followed by a final rotational test on day 49. Animals are euthanasized at this time,

in their induced (+ rapamycin) state, and processed for immunohistochemistry.

Behavioral rotational response to L-dopa

Behavioral analysis is performed using the classical rotational response to dopamine

agonists. In this case the agonist is dopamine synthesized from exogenous L-dopa, in the

unilateral 6-OHDA rat model of PD. Ungerstedt (1971) Acta Physiol. Scand. Suppl. 367: 69-

93. As shown in FIG. 4, three weeks after transduction, animals in the vector-infused (+)

rapamycin group show a robust contralateral turning response to L-dopa (5 mg/kg) that is

significantly higher than that of vector-infused (-) rapamycin group (215.13 ± 73.86 versus 16.33 ± 21.92 clockwise turns in 60 min, P < 0.001). Statistical differences are compared by

using one-way ANOVA analysis for multiple groups.

In the vector-infused (+) rapamycin group, the contralateral turning response to L-

dopa challenge at weeks 3, 5, and 7 is significantly increased with respect to pre-infusion

scores or time matched controls (vector-infused (-) rapamycin group and excipient-infused

(+) rapamycin group) (P < 0.001). In contrast, the vector-infused (+) rapamycin group is not

significantly different from the two control groups (P > 0.05) at the pre-infusion time point

and at time points following withdrawal of rapamycin (weeks 4 and 6).

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

theory, the gradual increase in rotational response over the course of seven weeks in both of

the control groups may be due to sensitization to repeated L-dopa treatment. Rotational response in the vector-infused (+) rapamycin group at the "off rapamycin

time points, weeks 4 and 6, is not significantly different from the vector-infused (-) rapamycin

group or the excipient-infused (+) rapamycin group at those time points, demonstrating the

reversibility of the induced response. The fact that induction of hAADC expression, and

subsequent decreased expression in untreated weeks, is observed over three successive

rapamycin treatment cycles strengthens the conclusion that rapamycin-induced induction of

gene expression in vivo is occurring.

Quantitation of hAADC transgene copies in infused striatum

Real-time quantitative PCR is performed on all rats to confirm that both of the vector-

infused groups are transduced with equivalent numbers of copies of hAADC gene. The

number of copies of the hAADC gene in the vector-infused (+) rapamycin group (222 ± 92

genome copies / 20 ng DNA) (± SD) is not different from the vector-infused (-) rapamycin

group (229 ± 48 genome copies / 20 ng DNA).

Immunohistochemistry and quantification of expression.

hAADC expression levels are evaluated by immunohistochemical analysis at seven

weeks post-infusion. A high magnification image within the infusion site of the striatum of a

representative animal from the vector-infused (+) rapamycin group is shown in FIG. 5 A, and

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

striatum. In contrast, rats that are infused with the vector but never induced (vector-infused (-

) rapamycin group) show a very low level of hAADC transgene expression (FIG. 5B). FIG. 6 shows low magnification images of AADC immunohistochemistry in whole

mounted brain sections from representative animals from each group at seven weeks

postintrastriatal infusion. Animals infused with AAV-CMV-TF + AAV-Z 12-hAADC (3 x 10¹⁰ vg of each vector) with rapamycin induction (A) or without rapamycin (B) are shown in

comparison to the excipient control with rapamycin (C). The left hemisphere is the site of

both 6-OHDA lesion and intrastriatal vector (or excipient) infusions. The right hemisphere is

unlesioned and uninfused, and thus staining of the right hemispheres reflects endogenous

staining from intact rat AADC-positive fibers.

6-OHDA lesioning causes depletion of the endogenous AADC, resulting in low

histochemical staining of the enzyme in the left hemisphere, as best illustrated in the

excipient-infused control group shown in FIG. 6C. Rats in the vector-infused (+) rapamycin

group all exhibit hAADC transgene staining on the infused left side (see, e.g., FIG. 6A). Rats

in the vector-infused (-) rapamycin group have low levels of hAADC transgene expression

(see, e.g., FIG. 6B). This hAADC expression is observed in five out of six vector-infused (-) rap animals. There are fewer positive cells, and lower intensity of staining per cell, in the

vector-infused (-) rapamycin group in comparison to the vector-infused (+) rapamycin group,

demonstrating only low level of hAADC expression from the inducible promoter in the

absence of rapamycin.

Quantitative stereology is also performed on serial brain sections obtained from

euthanized rats at seven weeks following intrastriatal infusion, immediately following

rapamycin dosing. AADC immunostaining is evaluated and quantitated by stereology in

serial sections of fixed brain tissue. An optical fractionator stereology protocol is used, an

efficient unbiased object counting method that is a combination of an optical dissector

protocol with statistically optimized spatial sampling procedures. Gundersen et al. (1988) Apmis 96:857-81; Gundersen (1986) J. Microsc. 143 (Pt l):3-45. Results are presented in Table 1, which reveals that the vector-infused (+) rapamycin group has the greatest anterior-

to-posterior distance of staining (3,720 ± 1,276 μm) (± SD), whereas there is a much lower

level of spread (1,920 ± 1,577 μm) in the vector-infused (-) rapamycin group.

TABLE l AADC Expression in Rat Brain

"n" is the number of hemispheres examined.

Data values are presented ± one standard deviation.

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

volume of spread within the striatum. The vector-infused (+) rapamycin group has the

greatest number of AADC-positive cells (75,825 +/- 30,506 cells), followed by a lower

number of positive cells (31,000 +/- 25,812 cells) in the vector-infused (-) rapamycin group

(P<0.01), and no detectable expression in the excipient-infused control group (data not

shown). Similarly, the vector infused (+) rapamycin group displays a large volume of

hAADC striatal spread (15.75 ± 8.16 mm³), whereas vector-infused (-) rapamycin group

exhibits a 55% lower volume of striatal spread (7.09 ± 5.69 mm³). Stereological analysis is a precise measure of the number of striatal neurons

transduced and the distribution of viral vector particles. However, total protein analysis is a

more accurate determination of the amount of hAADC enzyme produced because the amount

of hAADC created per cell may differ between groups. To confirm that the lower intensity of

hAADC expression in the uninduced group seen by immunostaining correlates with a lower

total level of hAADC expression, total protein is extracted from serial tissue sections and

examined by Western blot analysis.

A lower hAADC protein concentration in the uninduced group is confirmed. FIG. 7

shows images of the relevant gel bands, and a plot of integrated band intensities, from vector- infused (+/-) rapamycin and excipient-infused (+) rapamycin unilaterally 6-OHDA lesioned

rats showing changes in protein levels of hAADC (50 kDa) within the striatum. β-Actin is included as a loading control. AADC band density is significantly higher in the vector-

infused (+) rapamycin group in comparison to both control groups, PO.001. The band

intensities on Western blots from the vector-infused (+) rapamycin and (-) rapamycin rats are

102.04 ± 7.02 megapixels (MP) and 30.63 ± 3.47 MP, respectively, at seven weeks

postinfusion. The mean total AADC protein level is reduced by 88 %, after correction for the

presence of endogenous rat AADC levels, in the vector-infused (-) rapamycin group

compared to the vector-infused (+) rapamycin group. These data demonstrate a greater

difference in hAADC enzyme level between the two groups than that suggested by

stereological analysis.

Experimental details regarding experimental methods used in Example 2 are as

follows. Surgical procedures

6-OHDA-lesioned adult Sprague-Dawley rats (n = 6 rats for the excipient-infused and

the vector-infused (-) rapamycin groups and n = 8 rats for the vector-infused (+) rapamycin

group) are obtained from Taconic Farms (Germantown, NY). Rats are housed one per cage

under standard conditions: controlled temperature and humidity, 12-hour light cycle, and free

access to food and water. Vector is infused by convection-enhanced delivery (CED) to

achieve an optimal distribution throughout the striatum. Bankiewicz et al. (2000) Exp.

Neurol. 164:2-14; Lieberman et al. (1995) J. Neurosurg. 82:1021-9. Briefly, the vectors are

loaded into a polymer tubing (OD, 1/16"; ID, 0.030"; Upchurch Scientific, Oak Harbor, WA)

connected to a line filled with olive oil pumped from a 1 ml gas-tight Hamilton syringe.

Vector is delivered with a programmable pump (Bioanalytical Systems, hie, West Lafayette,

IN). The cannula, consisting of a fused silica capillary (OD, 164 μm; ID, 100 μm; Polymicro

Technologies, Phoenix, AZ) fitted into a 27-gauge needle, is connected to the distal end of the

polymer tubing. Anesthesia is induced with 3% isoflurane in O₂ flow (2 L/min) and animals

are placed in a stereotaxic frame (Kopf, Tujunga, CA). Anesthesia is then maintained with

1% isoflurane in O₂ through a mask fixed to the stereotaxic frame. Burr holes are drilled over

the target sites and cannulas are inserted vertically into the caudate-putamen at the following

coordinates relative to bregma and dura: AP 0.0 mm, ML -3.5 mm, DV -5.0 mm, with the

incisor bar set at -3.3 mm. Animals are unilaterally infused with 10 μl of a 1:1 ratio of the

two vectors at a rate of 0.5μl/min. At the end of the 20 min infusion period, the rate is

decreased to 0 μl/min for a 5 min rest and the cannula is slowly withdrawn.

Behavioral Analysis

Response to apomorphine (0.05 mg/kg, Sigma, St. Louis, MO) and L-dopa (methyl ester, Sigma) is evaluated with automated rotometers connected to a computer running

RotoMax rotational analysis software (AccuScan Instruments, Inc. Columbus, OH). Total

contralateral and ipsilateral turns are computed over 30 min (for apomorphine) and over 60

min (for L-dopa). Only rats with an average rotation > 6 contralateral turns/min for 30 min in

response to apomorphine (0.05 mg/kg) three weeks after the lesion are considered well

lesioned (Ungerstedt (1971) Acta Physiol. Scand. Suppl. 367:69-93) and tested for L-dopa

response. These animals are tested for L-dopa response using a dose below therapeutic range, 5 mg/kg of L-dopa methyl ester co-administered with 2.5 mg/kg of benserazide (Sigma, St.

Louis, Missouri). Over 95% of the rats tested do not rotate in response to 5 mg/kg L-dopa. Animals showing a net contralateral rotation to this dose are excluded from the experiment.

L-dopa response is evaluated before surgery and at different time points (3, 4, 5, 6, and 7

weeks) after the intrastriatal infusion.

Immunohistochemistry

For histological studies, animals are perfused through the aorta with saline, followed

by 4% paraformaldehyde (n = 6 rats for the excipient-infused and the vector-infused (-)

rapamycin groups and n = 8 rats for the vector-infused (+) rapamycin group). Brains are

postfixed overnight in 4% paraformaldehyde, equilibrated in graded sucrose solutions, and

frozen in isopentane. Brains are cut serially into 40-μm thick coronal sections on a cryostat.

Immunohistochemistry for AADC (Chemicon, Temecula, CA, 1:1500) is carried out on free-

floating sections. Sections are incubated in 3% hydrogen peroxide for 30 min to quench

endogenous peroxidases. After blocking for non-specific binding with 5% normal goat

serum, sections are incubated in primary antibody overnight at room temperature.

Incubations with a biotinylated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, 1:300) followed by streptavidin-conjugated horseradish peroxidase (Vector Laboratories,

1 :300) are carried out at room temperature, both for 1 h, and the complex is visualized with 3-

3'-diaminobenzidine (DAB, Vector Laboratories) and hydrogen peroxide. Sections are

mounted on gelatin-coated slides, dried, dehydrated in ascending ethanol series, cleared in

xylenes, and mounted using Cytoseal-60 (Richard- Allen Scientific, Kalamazoo, MI).

Anterior-to-posterior distribution of hAADC immunostaining is determined by the formula (n

x 12 x 40μm) where n is the number of sections with hAADC positive cells, 40μm is the

thickness of the section, and every twelfth section is examined. The volume of distribution

and positive cell count were estimated in serial sections (every twelfth), stained for AADC using the Optical Fractionator-Optical Dissector design-based stereology method under 63X

magnification on a Zeiss microscope equipped with a video camera and Stereoinvestigator

stereology software (Microbrightfield, Williston, VT). CEE < 5% for each group. Results

are reported as mean ± SD. Student's t-test is used to measure statistical significance.

Real-time quantitative PCR

The vector AAV-Z12-hAADC used in this study contains the human AADC target

gene. The Q-PCR primers and probe anneal to exons 2 and 3 of the AADC gene, thus

spanning an intron not present in the vector sequence and thereby minimizing amplification

of genomic DNA. Real-time Q-PCR (Heid et al. (1996) Genome Res. 6:986-94) is

standardized with plasmid DNA containing the vector insert. The plasmid is linearized with a

restriction enzyme, purified, quantified by UV absorbance, and diluted in Q-PCR dilution

buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 μg/ml yeast tRNA, and 0.1% Tween 80)

to give 10 standards ranging from three to 10⁶ copies per reaction. Each standard is run in

three replicate 50 μl reactions in a 96-well optical plate. 10 μl of sample, each containing 20 ng of DNA, is added to three replicate Q-PCR wells containing 40 μl reaction mixture. PCR

is carried out on an Applied Biosystems 7700 Sequence Detection System. The number of

hAADC gene copies is calculated by comparison to the standard curve, and multiplying the

resulting copies per well by two, assuming that one copy of double-stranded plasmid DNA is

equivalent to two single-stranded vector genomes.

Western Blot Analysis

Ten serial sections (40 μm each) of whole brains are homogenized separately with a

hand-held homogenizer in a lysis buffer containing a mixture of phosphatase inhibitors and

proteinase inhibitors. Protein is quantified using the Bradford method. Protein samples (15

μg) are separated on SDS-PAGE gel (4-15% gradient gel; Bio-Rad, Hercules, California) and

transferred to polyvinylidene difluoride filters (Millipore, Bedford, Massachusetts).

The filters are blocked with 3% milk and incubated for 1 hr with a polyclonal rabbit

anti-AADC (1 :500 Chemicon, Temecula, California) primary antibody. Then the blots are

incubated for 1 hr at room temperature (RT) with a corresponding HRP-conjugated secondary

antibody (1:3000; Amersham Biosciences, Arlington Heights, Illinois), visualized in ECL

solution (PerkinElmer Life Sciences, Emeryville, California) for 1 min, an exposed onto X-

Omat film from Kodak (Rochester, New York) for 1—30 min. Finally, the blots are incubated

in a stripping buffer (67.5 mM Tris, pH 6.8, 2% SDS, and 0.7% β-mercaptoethanol) for

30 min at 50°C and reprobed with a polyclonal rabbit anti-β-actin antibody (1 : 1000; Alpha

Diagnostics, San Antonio, Texas) as loading controls. The anti-AADC primary antibody has

been used extensively in previous studies, and Western blot bands observed in this study

show the same band size (-50 kDa) as indicated in the antibody information sheet. The density of each specific band is measured with a computer-assisted imaging

analysis system (Alphalmager , Alpha Innotech Corporation, San Leandro, California).

There is no significant difference in the density of β-actin-loading control bands among

groups. To compare the differences between the excipient-infused (+) rapamycin control and

vector-infused groups, the density of each specific band is first normalized against the density of the corresponding internal loading band (n = 3 for each group). The percent reduction in

total protein is determined after subtracting the endogenous hAADC level found in the

excipient-infused rats from both vector-infused groups. Differences are compared by using

one-way ANOVA for multiple groups.

EXAMPLE 3

REGULATED EXPRESSION OF GDNF IN VITRO

Mammalian cells are transduced with a dimerizer-regulatable GDNF transgene

construct as follows.

AAV-GDNF Vectors

FIGS. 8 A and 8B are diagrams of recombinant AAV vector plasmid constructs for

delivery of a regulatable human GDNF (hGDNF) transgene. A control vector for delivery of

constitutively expressed hGDNF (pAAV-CMV-hGDNF) is shown at FIG. 8C.

The regulatable constructs (FIGS. 8 A and 8B) involve a single rAAV vector carrying

the genes encoding hGDNF and the activation domain fusion protein and the DNA binding

domain fusion protein components of the transcription factor. In both constructs expression

of hGDNF is driven by a minimal IL-2 promoter adjacent to eight binding sites for the dimerizable transcription factor described in greater detail below. In the construct illustrated at FIG. 8A (ρAAV-TF-Z8-hGDNF), a CMV

enhancer/promoter drives expression a single transcript encoding both the activation and

DNA binding domains of the transcription factor, with an internal ribosome entry site (IRES)

between the two. In the construct illustrated at FIG. 8B, in contrast, an SV40 promoter drives

expression of the DNA binding domain and a CMV enhancer/promoter drives expression of

the activation domain, on a different transcript, from the opposite strand (i.e. in the opposite

direction).

The DNA binding domain fusion protein contains two DNA binding domains from

the human transcription factor Zif268, a homeodomain derived from Oct-1 (ZFHDl), and

three drug binding domains from the cytosolic receptor for FK506 (3xFKBP).

The activation domain fusion protein contains the rapamycin binding domain of

human FRAP (FRB*) fused to the transcriptional activation domain derived from the p65

subunit of NFKB (p65). The FRB* domain illustrated in FIGS. 8A and 8B is described in

Example 1.

AAV2 inverted terminal repeat (ITR), minimal SV40 polyadenylation (Min. SV40

pA), minimal rabbit β-globin polyadenylation (Min. RBG pA) and minimal human growth

hormone polyadenylation (Min hGH pA) sequences are indicated.

In control vector pAAV-CMV-hGDNF (FIG. 8C) the CMV promoter/enhancer drives

expression of hGDNF. AA V2 inverted terminal repeat (ITR) and human growth hormone

polyadenylation (pA) sequences are indicated.

The results of the rapamycin-induction experiment are presented at FIG. 9, in which

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

GDNF expression irrespective of rapamycin treatment.

Experimental details regarding experimental ELISA assay methods used in Example 3

are as follows.

GDNFELISA

ELISA assays to quantify GDNF expression are performed as follows.

HEK-293 cells (5x10⁵ cells/well) are grown over night in two 6-well plates to reach

60-70% confluence. Plates are transfected with 10 μg of either pAAV-TF-Z8-hGDNF or pAAV-CMV-hGDNF using 300μM CaCl₂. Six hours later the media are exchanged with

fresh media containing rapamycin (0 nM, 5 nM, or 25 nM, each in duplicate) and grown for

three days. Both media and cells are harvested separately, snap frozen and stored in -8O⁰C

until ELISA is performed. All samples are acid treated with IN HCl to below pH 3.0 for

15min., and then neutralized back to about pH 7.6 with IN NaOH.

Media samples are assayed for the presence of GDNF using the Promega E_max®

ImmunoAssay system (Promega, Inc., Madison, Wisconsin). Coating buffer is added to the

96-well plate, which is incubated overnight at 4⁰C. The coating buffer is removed and the

plate is drained. Blocking buffer (200 μL) is added to the plate and incubated for one hour at

room temperature without shaking. The blocking buffer is removed and the plate is drained.

Two 8-well columns of the 96-well plate are designated for GDNF standards. IX

Block & Sample Buffer (100 μL/well) is added to rows B-H of the standards columns.

Diluted GDNF standard (200 μL of 1000 pg/ml) is added to row A of the standard columns,

and a 2-fold serial dilution of 100 μL /well is performed down to row G. Row H is a buffer-

only control with no GDNF. Experimental samples are diluted to 1:300 for pAAV-TF-Z8-hGDNF and 1 :1000 for

pAAV-CMV-hGDNF experiments, and 100 μL of each is added to duplicate wells and

incubated for six hours at room temperature with shaking (500 rpm). Wells are washed five

times, each time with approximately 400 μL of the recommended wash buffer from the kit.

100 μL of a 1:500 dilution of anti-hGDNF polyclonal antibody (in IX Block & Sample

Buffer) is added to each well and incubated overnight at 4⁰C without shaking. The plate is

washed again as described above. 100 μL of a 1:250 dilution of anti-chicken IgY, HRP

conjugate (in IX Block & Sample Buffer) is added to each well and incubated for two hours

at room temperature with shaking (500 rpm). The plate is washed again as described above.

100 μL of room temperature TMB One solution (containing the HRP substrate 3,3',5,5'-

tetramethylbenzidine) is added to each well and incubated at room temperature for 15 minutes

without shaking. The color development is stopped by adding 100 μL of IN hydrochloric

acid to each well. The absorbance is recorded at 450 nm on a plate reader within 30 minutes

of addition of TMB. GDNF levels in pg/ml are determined by comparison of signals obtained from experimental samples with the GDNF standards on the same plate. The standard

deviation is derived from a total of four determinations for each sample (duplicate samples on

each of two plates).

While preferred illustrative embodiments of the present invention are described, it will

be apparent to one skilled in the art that various changes and modifications may be made

therein without departing from the invention, and it is intended in the appended claims to

cover all such changes and modifications that fall within the true spirit and scope of the

invention. All publications, patents, patent applications, sequences and database entries referred

n are hereby incorporated by reference in there entireties.

Claims

We claim:

1. A pharmaceutical composition for the treatment of a subject with a neurological

disorder, comprising: a recombinant adeno-associated virus (AAV) vector encoding a regulatable

transcription factor having transcription enhancement activity; and

a recombinant AAV vector encoding a transgene;

wherein expression of the transgene is affected by the activity of the transcription

factor.

2. The pharmaceutical composition of claim 1 , wherein the regulatable transcription

factor and the transgene are encoded on separate rAAV vectors.

3. The pharmaceutical composition of either of claims 1 -2, wherein the activity of the

regulatable transcription factor is increased in the presence of rapamycin or a rapamycin

analog.

4. The pharmaceutical composition of claim 3, wherein the rapamycin analog is

AP21967.

5. The pharmaceutical composition of any of claims 1 -4, wherein the neurological

disorder is Parkinson's disease.

6. The pharmaceutical composition of any of claims 1 -5, wherein the transgene is

aromatic L-amino acid decarboxylase (AADC).

7. The pharmaceutical composition of any of claims 1-5, wherein the transgene is glial

cell line-derived neurotrophic factor (GDNF).

8. A method of treating a subject with a neurological disorder, comprising administering

a therapeutically effective dose of the pharmaceutical composition of any of claims 1-7.

9. A kit for performing the method of claim 8, comprising:

a recombinant AAV vector encoding a regulatable transcription factor;

a recombinant AAV vector encoding a transgene; and

rapamycin or a rapamycin analog.

10. Use of a composition according to any of claims 1 -7 in a method of treating a subj ect

with a neurological disorder.

11. Use of a recombinant AAV vector encoding a transgene and a recombinant AAV

vector encoding a regulatable transcription factor having transcription enhancement activity,

wherein expression of the transgene is affected by the activity of the transcription factor, in

the manufacture of a composition for treatment of a subj ect with a neurological disorder.