Methods of making and using recombinant AAV vectors and virions for gene delivery to the lung are described. The recombinant AAV virions are derived from caprine AAV and bovine AAV, both of which display tropism for lung tissue.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is based on the discovery of novel AAV variants with tropism to lung tissue. The AAV variants are derived from a caprine source and from a bovine source. The rAAV virions made using caprine and bovine AAV sequences are able to efficiently transduce lung cells and tissues and are especially useful for delivering heterologous nucleic acid molecules (HNAs) to subjects with lung disorders. Moreover, the caprine and bovine AAV sequences display significantly decreased immunoreactivity relative to rAAV virions typically used in gene therapy studies. Thus, subjects that have been previously exposed to AAV, either by natural infection or due to previous gene therapy or nucleic acid immunization treatments, and have therefore developed anti-AAV antibodies, can still benefit from the use of the caprine and bovine rAAV virions of the invention. Moreover, the caprine and bovine rAAV virions described herein are readily administered and display efficient transduction using a vascular route of administration. Thus delivery can be achieved by simple intravenous administration rather than via the airway, which is often highly damaged in serious lung disorders.

The rAAV virions described herein are therefore useful for treating or preventing a wide variety of lung disorders in vertebrate subjects in need of such treatment, whether or not the subject has been previously exposed to any of the various AAV serotypes.

In one aspect, the present invention provides methods and AAV vectors for the efficient delivery of HNAs, such as a gene of interest, to the lung cells or tissue of a vertebrate subject, such as a human, to provide a therapeutic effect. In certain preferred embodiments, rAAV virions are derived from caprine AAV In other rAAV virions are derived from bovine AAV. In some embodiments the rAAV virions comprise an HNA encoding antisense RNA, ribozymes, or one or more genes of interest that express proteins. The rAAV virions can be used to deliver these HNAs to the lung, wherein expression of said antisense RNA, ribozymes, or genes in lung cells or tissue provides for a biological effect in a mammalian subject.

Thus, in one aspect, the invention is directed to a method for delivering HNAs to the lung of a vertebrate subject using an rAAV virion. In one embodiment, the method comprises (a) providing a caprine rAAV virion, wherein the virion comprises a heterologous nucleic acid molecule encoding a therapeutic protein operably linked to control elements capable of directing the in vivo transcription and translation of said protein; and (b) delivering the recombinant AAV virion to the vertebrate subject, whereby the protein is expressed in the lung at a level that provides a therapeutic effect. In another embodiment, a bovine rAAV virion is used instead of the caprine rAAV in the method of the previous sentence.

In another embodiment, the rAAV virions containing an HNA are injected directly into a muscle (e.g., cardiac, smooth and/or skeletal muscle). In another embodiment the rAAV virions containing an HNA are administered into the vasculature via injection into veins, arteries, or other vascular conduits, or by using techniques such as isolated limb perfusion.

In an additional embodiment, the virions contain a gene encoding CFTR, the gene deficient or missing in cystic fibrosis patients, that when expressed at a sufficient concentration provides for a therapeutic effect, such as amelioration or reduction of symptoms caused by cystic fibrosis.

In another embodiment the virions contain a gene encoding alpha-1-antitrypsin that when expressed at a sufficient concentration provides for a therapeutic effect, such as amelioration or reduction of symptoms caused by alpha-1-antitrypsin deficiency (e.g. emphysema).

In another embodiment, the virions contain a gene encoding an enzyme capable of removing toxic metabolites that tend to accumulate in diseased lung tissue (e.g. such as superoxide dismutase (SOD) and catalase) that when expressed at a sufficient concentration, provides for a therapeutic effect, such as amelioration or reduction of the toxic metabolites.

In yet a further embodiment, the virions contain a gene encoding an anti-tumor agent or a tumor suppressor that when expressed at a sufficient concentration provides for a therapeutic effect, such as a reduction in tumor size and/or growth. Such agents include immunomodulators, such as any of the various cytokines including interleukin-1, interleukin-2, interleukin-3, interleukin-4, and gamma-interferon; p53; the retinoblastoma (rb) gene product; antisense oncogenes, e.g., anti-c-myc and anti-k-ras; and other growth control-related genes for cancer gene therapy.

In additional embodiments, the virions contain a gene encoding an agent useful for treating primary pulmonary hypertension, pulmonary vascular disease secondarily associated with chronic airways obstruction, and connective tissue diseases.

In further embodiments, the invention is directed to a recombinant AAV virion comprising any of the HNAs described above.

In yet further embodiments, the invention is directed to a method of delivering an HNA to a lung cell or tissue of a vertebrate subject using a recombinant caprine or bovine AAV virion. The method comprises:

(a) providing a recombinant caprine or bovine AAV virion as above, wherein the virion comprises an HNA encoding a protein;

(b) administering the recombinant AAV virion to the subject, whereby the protein is expressed at a level in lung cells or tissue that provides a therapeutic effect.

In certain embodiments, the recombinant AAV virion is delivered by intramuscular injection, or into the bloodstream (e.g. intravenously or intraarterially).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell Scientific Publications); 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 (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

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 particular formulations 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.

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.

Central to the present invention is the discovery of novel caprine and bovine AAV sequences useful in the production of rAAV virions that display remarkable lung tropism. Thus, AAV vectors and virions derived from these caprine and bovine AAV sequences are useful for targeting gene delivery to lung cells and tissue. Delivery of genes to the pulmonary vascular endothelium is a rational approach for therapy of pulmonary vascular diseases including, but not limited to, primary pulmonary hypertension, pulmonary vascular disease secondarily associated with chronic airways obstruction, connective tissue diseases, HIV infection, and emphysema. Furthermore, in view of the exposure of this vascular bed to the entire cardiac output, delivery to the lungs is useful to achieve systhemic delivery of secreted factors. Aside from vascular disease per se, the pulmonary bed is frequently the site of metastatic spread of malignancy, and once this occurs conventional therapy options are most often inadequate.

Thus, the vectors and virions find use in the treatment of a wide variety of lung disorders, including without limitation, cystic fibrosis (CF), alpha-1-antitrypsin gene deficiencies, lung cancer, lung deterioration caused by degenerative lung diseases, and pulmonary vascular disease.

For example, it is known that CF is caused by a mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Thus rAAV virions containing a gene encoding CFTR can be administered to patients suffering from CF to provide for a therapeutic effect, such as amelioration or reduction of symptoms caused by cystic fibrosis.

Similarly, a common type of alpha-1-antitrypsin deficiency, termed protease inhibitor type Z (PiZ), is caused by a single nucleotide substitution that results in a single amino acid substitution (glutamate 342 to lysine). The replacement of glutamate 342 with a lysine apparently prevents normal folding of the protein. Thus, rAAV virions containing a gene encoding the wild-type alpha-1-antitrypsin can be administered to patients suffering from alpha-1-antitrypsin to provide for a therapeutic effect, such as amelioration or reduction of symptoms caused by alpha-1-antitrypsin deficiency, such as emphysema.

Additionally, the rAAV vectors and virions of the present invention can also be used to deliver genes to patients with degenerative diseases of the lung, e.g., genes encoding enzymes capable of removing toxic metabolites which tend to accumulate in diseased lung tissue, such as superoxide dismutase (SOD) and catalase.

The caprine and bovine AAV vectors and virions of the present invention are also useful for treating cancers of the lung. For example, the caprine and bovine AAV vectors and virions can be used to deliver a gene encoding an anti-tumor agent or a tumor suppressor, that when expressed at a sufficient concentration, provides for a therapeutic effect, such as a reduction in tumor size and/or growth. Such agents include immunomodulators, such as any of the various cytokines including interleukin- 1, interleukin-2, interleukin-3, interleukin-4, and gamma-interferon; p53; the retinoblastoma (rb) gene; antisense oncogenes, e.g., anti-c-myc and anti-k-ras; and other growth control-related genes for cancer gene therapy.

Moreover, because the pulmonary vasculature bed is exposed to the entire cardiac output, delivery of a gene of interest to the lungs is useful to achieve systhemic delivery of a large number of secreted factors, including but not limited to, one or more peptides, polypeptides, or proteins that are useful for the treatment or prevention of disease states in a mammalian subject. Such DNA and associated disease states include, but are not limited to: DNA encoding glucose-6-phosphatase, associated with glycogen storage deficiency type 1A; DNA encoding phosphoenolpyruvate-carboxykinase, associated with Pepck deficiency; DNA encoding galactose-1 phosphate uridyl transferase, associated with galactosemia; DNA encoding phenylalanine hydroxylase, associated with phenylketonuria; DNA encoding branched chain alpha-ketoacid dehydrogenase, associated with Maple syrup urine disease; DNA encoding fumarylacetoacetate hydrolase, associated with tyrosinemia type 1; DNA encoding methylmalonyl-CoA mutase, associated with methylmalonic acidemia; DNA encoding medium chain acyl CoA dehydrogenase, associated with medium chain acetyl CoA deficiency; DNA encoding omithine transcarbamylase, associated with omithine transcarbamylase deficiency; DNA encoding argininosuccinic acid synthetase, associated with citrullinemia; DNA encoding low density lipoprotein receptor protein, associated with familial hypercholesterolemia; DNA encoding UDP-glucouronosyltransferase, associated with Crigler-Najjar disease; DNA encoding adenosine deaminase, associated with severe combined immunodeficiency disease; DNA encoding hypoxanthine guanine phosphoribosyl transferase, associated with Gout and Lesch-Nyan syndrome; DNA encoding biotinidase, associated with biotinidase deficiency; DNA encoding beta-glucocerebrosidase, associated with Gaucher disease; DNA encoding beta-glucuronidase, associated with Sly syndrome; DNA encoding peroxisome membrane protein 70 kDa, associated with Zellweger syndrome; DNA encoding porphobilinogen deaminase, associated with acute intermittent porphyria; DNA encoding erythropoietin for treatment of anemia due to thalassemia or to renal failure; DNA encoding vascular endothelial growth factor, DNA encoding angiopoietin-1, and DNA encoding fibroblast growth factor for the treatment of ischemic diseases; DNA encoding thrombomodulin and tissue factor pathway inhibitor for the treatment of occluded blood vessels as seen in, for example, atherosclerosis, thrombosis, or embolisms; DNA encoding aromatic amino acid decarboxylase (AADC), and DNA encoding tyrosine hydroxylase (TH) for the treatment of Parkinson's disease; DNA encoding the beta adrenergic receptor, DNA encoding anti-sense to, or DNA encoding a mutant form of, phospholamban, DNA encoding the sarco(endo)plasmic reticulum adenosine triphosphatase-2 (SERCA2), and DNA encoding the cardiac adenylyl cyclase for the treatment of congestive heart failure; DNA encoding a tumor suppressor gene such as p53 for the treatment of various cancers, including lung cancer; DNA encoding a cytokine such as one of the various interleukins for the treatment of inflammatory and immune disorders and cancers; DNA encoding dystrophin or minidystrophin and DNA encoding utrophin or miniutrophin for the treatment of muscular dystrophies; and, DNA encoding insulin for the treatment of diabetes.

The invention also provides caprine and bovine rAAV virions comprising a gene or genes coding for blood coagulation proteins, which proteins may be delivered, using the methods of the present invention, to the lung of a mammal having hemophilia for the treatment of hemophilia. Thus, the invention includes: delivery of the Factor IX gene to a mammal for treatment of hemophilia B, delivery of the Factor VIII gene to a mammal for treatment of hemophilia A, delivery of the Factor VII gene for treatment of Factor VII deficiency, delivery of the Factor X gene for treatment of Factor X deficiency, delivery of the Factor XI gene for treatment of Factor XI deficiency, delivery of the Factor XIII gene for treatment of Factor XIII deficiency, and, delivery of the Protein C gene for treatment of Protein C deficiency. Delivery of each of the above-recited genes to the cells of a mammal is accomplished by first generating a caprine and bovine rAAV virion comprising the gene and then administering the rAAV virion to the mammal. Thus, the invention includes rAAV virions comprising genes encoding any one of Factor IX, Factor VIII, Factor X, Factor VII, Factor XI, Factor XIII or Protein C.

Generally, caprine rAAV virions will include at least a caprine AAV capsid protein, and the bovine rAAV virions will include at least a bovine AAV capsid protein. Thus, by "caprine rAAV virion" is meant an rAAV virion that includes at least one caprine capsid protein, and by "bovine rAAV virion" is meant an rAAV virion that includes at least one bovine capsid protein. As explained above, the AAV cap region encodes at least three proteins: VP1, VP2, and VP3. FIG. 1 (see Original Patent) shows the overlapping structure of this region from AAV-2 which is similar to the caprine structure (see further below). Preferably, the rAAV virions include at least the caprine VP3 region, but can include the entire VP2 or VP1 region. The caprine AAV VP1 sequence is highly homologous to the VP1 sequence of AAV-5, but is approximately 100 times more resistant to neutralization by existing AAV antibodies than the native AAV-5 sequence.

More particularly, a 2805 bp PCR fragment of the caprine AAV described herein, encoding 603 bp of rep, the central intron, and all of cap, shows 94% homology to the corresponding AAV-5 sequence. The DNA and protein homologies for the partial rep are 98% and 99%, respectively. A comparison of the caprine VP1 coding sequence with a primate AAV-5 VP1 coding sequence is shown in FIGS. 2A-2B (see Original Patent). The DNA for the cap region of the caprine AAV is 93% homologous to that of AAV-5. The amino acid sequences for the caprine VP1 versus a primate AAV-5 is shown in FIG. 3 (see Original Patent). The caprine sequence encodes a VP1 protein of 726 amino acids, while AAV-5 VP1 is 724 amino acids in length. Additionally, the sequences display 94% sequence identity and 96% sequence similarity. There are 43 amino acid differences between the caprine and the primate AAV-5 VP1 sequence. With respect to the linear amino acid sequence of VP1, the distribution of the amino acid differences between AAV-5 and caprine AAV is highly polar. All of the amino acid differences occur exclusively in the C-terminal hypervariable region of VP1 in a scattered fashion. This region relative to AAV-5 and caprine includes approximately 348 amino acids from amino acid 386 to the C-terminus, numbered relative to AAV-5 VP1.

Moreover, rAAV virions including such caprine sequences show substantial tropism to lung tissue. Thus, the present invention involves the production and use of caprine AAV sequences for incorporation into rAAV virions. Such rAAV virions can be used to deliver a "heterologous nucleic acid" (an "HNA") to the lung tissue of a vertebrate subject, such as a mammal. As explained above, a "recombinant AAV virion" or "rAAV virion" is an infectious virus composed of an AAV protein shell (i.e., a capsid), derived from caprine AAV, encapsulating a "recombinant AAV (rAAV) vector," the rAAV vector comprising the HNA and one or more AAV inverted terminal repeats (ITRs). AAV vectors can be constructed using recombinant techniques that are known in the art and include one or more HNAs flanked by functional ITRs. The ITRs of the rAAV vector need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion, or substitution of nucleotides, so long as the sequences provide for proper function, i.e., rescue, replication, and packaging of the AAV genome.

Recombinant AAV virions may be produced using a variety of techniques known in the art, including the triple transfection method (described in detail in U.S. Pat. No. 6,001,650, the entirety of which is incorporated herein by reference). This system involves the use of three vectors for rAAV virion production, including an AAV helper function vector, an accessory function vector, and a rAAV vector that contains the HNA. One of skill in the art will appreciate, however, that the nucleic acid sequences encoded by these vectors can be provided on two or more vectors in various combinations. As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., that is capable of replication when associated with the proper control elements and that can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

The AAV helper function vector encodes the "AAV helper function" sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Examples of vectors suitable for use with the present invention include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, the disclosure of which is hereby incorporated by reference in its entirety.

The accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (i.e., "accessory functions"). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus. In a preferred embodiment, the accessory function plasmid pladeno5 is used (details regarding pLadeno5 are described in U.S. Pat. No. 6,004,797, the disclosure of which is hereby incorporated by reference in its entirety). This plasmid provides a complete set of adenovirus accessory functions for AAV vector production, but lacks the components necessary to form replication-competent adenovirus.

The rAAV vector containing the heterologous nucleic acid (HNA) may be constructed using ITRs from any of the various AAV serotypes. The HNA comprises nucleic acid sequences joined together that are otherwise not found together in nature, this concept defining the term "heterologous." To illustrate the point, an example of an HNA is a gene flanked by nucleotide sequences not found in association with that gene in nature. Another example of an HNA is a gene that itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to HNAs, as used herein. An HNA can comprise an anti-sense RNA molecule, a ribozyme, or a gene encoding a polypeptide.

The HNA is operably linked to a heterologous promoter (constitutive, cell-specific, or inducible) such that the HNA is capable of being expressed in the patient's target cells (here lung) under appropriate or desirable conditions. Numerous examples of constitutive, cell-specific, and inducible promoters are known in the art, and one of skill could readily select a promoter for a specific intended use, e.g., the selection of the constitutive CMV promoter for strong levels of continuous or near-continuous expression, or the selection of the inducible ecdysone promoter for induced expression. Induced expression allows the skilled artisan to control the amount of protein that is synthesized. In this manner, it is possible to vary the concentration of therapeutic product. Other examples of well known inducible promoters are: steroid promoters (e.g., estrogen and androgen promoters) and metallothionein promoters.

As explained above, in one embodiment, the invention relates to novel AAV virions including caprine and bovine capsids and comprising HNAs coding for one or more anti-sense RNA molecules, the rAAV virions preferably administered to one or more muscle cells or tissue of a mammal. Antisense RNA molecules suitable for use with the present invention in cancer anti-sense therapy or treatment of viral diseases have been described in the art. See, e.g., Han et al., (1991) Proc. Natl. Acad. Sci. USA 88:4313-4317; Uhlmann et al., (1990) Chem. Rev. 90:543-584; Helene et al., (1990) Biochim. Biophys. Acta. 1049:99-125; Agarawal et al., (1988) Proc. Natl. Acad. Sci. USA 85:7079-7083; and Heikkila et al., (1987) Nature 328:445-449. The invention also encompasses the delivery of ribozymes using the methods disclosed herein. For a discussion of suitable ribozymes, see, e.g., Cech et al., (1992) J. Biol. Chem. 267:17479-17482 and U.S. Pat. No. 5,225,347.

Preferably, the rAAV virions comprise HNAs coding for one or more polypeptides for treating a lung disorder. The rAAV virions are preferably administered to one or more cells or tissue of a mammal, and display tropism for lung. Thus, the invention embraces the delivery of HNAs encoding one or more peptides, polypeptides, or proteins, that are useful for the treatment or prevention of lung disease states in a mammalian subject. Such DNA and associated disease states are described above.

Delivery of the recombinant virions containing one or more HNAs to a mammalian subject may be by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. The recombinant caprine and bovine virions of the invention may also be introduced into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403 and herein incorporated by reference, can also be employed by the skilled artisan to administer the virions into the vasculature of an isolated limb to potentially enhance transduction into lung cells or tissue.

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), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene (or anti-sense RNA or ribozyme) expression required to achieve a therapeutic effect, the specific disease or disorder being treated, a host immune response to the rAAV virion, a host immune response to the gene (or anti-sense RNA or ribozyme) expression product, and the stability of the gene (or anti-sense RNA or ribozyme) product. One of skill in the art can readily determine a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.

Generally speaking, by "therapeutic effect" is meant a level of expression of one or more HNAs sufficient to alter a component of a lung disease (or disorder) toward a desired outcome or clinical endpoint, such that a patient's disease or disorder shows clinical improvement, often reflected by the amelioration of a clinical sign or symptom relating to the disease or disorder.
 

Claim 1 of 4 Claims

1. An infectious recombinant adeno-associated virus (AAV) virion comprising: an AAV vector comprising a heterologous nucleic acid molecule encoding a protein, wherein the nucleic acid molecule is flanked on each end with an AAV inverted terminal repeat, wherein the AAV vector is encapsidated by a viral capsid comprising caprine VP1.

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