WO2020039425A1

WO2020039425A1 - Vectors expressing klotho for treating cancer

Info

Publication number: WO2020039425A1
Application number: PCT/IL2019/050913
Authority: WO
Inventors: Carmela ABRAHAM; Menachem Abraham; Assumpcio BOSCH; Miguel CHILLON; Tamar Rubinek; Ido Wolf
Original assignee: Klogenix Llc; The Medical Research, Infrastructure And Health Services Fund Of The Tel-Aviv Medical Center; Universitat Autonoma De Barcelona; Institut Catala De Recerca I Estudis Avancats (Icrea)
Priority date: 2018-08-21
Filing date: 2019-08-14
Publication date: 2020-02-27

Abstract

Compositions and methods for inhibiting tumor growth in subjects in need thereof are provided, utilizing gene transfer vectors, such as viral vectors, comprising a nucleotide sequence encoding a klotho protein operably linked to at least one regulatory sequence directing its expression.

Description

VECTORS EXPRESSING KLOTHO FOR TREATING CANCER

FIELD OF THE INVENTION

The present invention relates to the treatment of cancer using gene therapy. In particular, the present invention relates to the treatment of cancer using gene transfer vectors, e.g., viral vectors, expressing klotho proteins.

BACKGROUND OF THE INVENTION

Klotho is a protein known to have aging suppressor effects, primarily expressed in the kidneys and brain but also in other organs such as ovaries, testes, placenta and pituitary. Klotho exists in both membrane-bound and soluble forms, which exert numerous distinct functions.

Membrane-bound klotho is a single-pass transmembrane protein with an N-terminal signal sequence, an extracellular domain composed of two homologous regions, termed KL1 and KL2, a transmembrane domain and a short intracellular domain (see

illustration and table in Figure 1). The extracellular domain of klotho contains two recognition sites of metalloproteinases, one between KL2 and the transmembrane domain and another between KL1 and KL2. Cleavage by the metalloproteinases produces soluble klotho proteins that either contain KL1, KL2, or both. It appears that the predominant soluble klotho protein that is shed from the membrane is the one containing both KL1 and KL2. Finally, an alternatively spliced, secreted, form of klotho has been identified, containing the KL1 domain with a tail of additional 15 amino acids at the C-terminus. The secreted form is abundant mainly in the brain.

Klotho was found to play a role, inter alia, in phosphate homeostasis, calcium homeostasis and suppression of insulin/IGF- 1 signaling. Accumulating data indicate a role for reduced klotho levels in chronic kidney disease.

Klotho was also found to be a tumor suppressor. Extensive research identified relevant pathways such as inhibition of TGFpi, Wnt, FGF2 and IGF1 signaling, and investigated structure-activity relationship (SAR) in several diseases including cancer, by studying the membrane bound as well as the soluble forms of klotho. In a meta-analysis study, it was shown that klotho protein expression is associated with a lower risk and progression of malignancies, and that klotho may be a protective factor against malignancies risk/progression.

In the vast majority of cancers, klotho expression is lower than in healthy cells, with the gene promoter being typically hypermethylated and/or the histone being deacetylated.

Tumor suppression has been demonstrated in animal studies using systemic supplementation with recombinant klotho.

US 9,987,326 and US 2012/0178699, to some of the inventors of the present invention, disclose klotho protein or DNA encoding klotho protein for use in the treatment of cancer. No specific gene therapy vectors are disclosed or suggested, and no anti-tumor activity using gene therapy vectors is demonstrated.

Masso et al. (2017) Mol Psychiatry, 1-11, studied the role of s-KL in cognitive processes. It was hypothesized that it is a neuroprotective protein involved in the onset and / or progression of cognitive deficits associated with aging. To explore its effects, s-KL levels in the brains of adult wild-type C57B1/6J male mice were modified using intraventricular or hippocampal administration of AAVrhlO gene therapy vectors.

WO 2017/085317, to some of the inventors of the present invention, discloses using secreted splicing variant of mammal klotho (s- KL) as an agent for the prevention and/or treatment of cognitive and/or behavior impairments. Also disclosed are gene constructs and expression vectors useful in gene therapy for the delivery of said s-KL variant to the central nervous system (CNS) of a mammal, in particular a rodent or a human. Pharmaceutical compositions comprising either the protein s-KL or any gene construct for expressing the protein in the CNS are also disclosed.

Despite the potential of using klotho as a tumor suppressor in the treatment of cancer, to date, the biopharma industry has been unsuccessful in consistent production of klotho at the appropriate quantity and quality that is required for clinical purposes, and no klotho-based treatment has reached clinical trials. The industry’s view is that this is due to difficulties in manufacturing recombinant klotho proteins in the appropriate quantity and quality, and in addition, due to challenges in developing novel effective small molecules that up-regulate klotho.

Improved compositions and methods based on klotho for the treatment of cancer are highly desired. SUMMARY OF THE INVENTION

The present invention provides, according to some aspects, compositions and methods for inhibiting tumor growth in subjects in need thereof using recombinant vectors, such as recombinant viral vectors, expressing klotho proteins.

The present invention is based in part on the finding that a single administration of a viral vector encoding a klotho protein successfully and significantly inhibited the growth of tumors in a mouse model. As exemplified hereinbelow, the single administration of the klotho vector to tumor-bearing mice led to tumors with significantly reduced size at the end of the study compared to treatment with a control vector, indicating that growth of the tumors was significantly inhibited in mice that received the klotho vector. It is noted that the vectors were administered 10 days following tumor inoculation, when tumors were already established in the mice, further strengthening the effect seen with the klotho vector in treating tumors and suppressing their growth.

The present invention demonstrates for the first time the production of functional klotho using gene therapy, at clinically-relevant amounts, that are capable of achieving a significant therapeutic outcome. Despite past reports linking klotho and anti-tumor effects, such a dramatic tumor reduction has not been seen in animal models in vivo.

The present invention enables consistent production of klotho in vivo at clinically- relevant quantity and quality, thus overcoming a major obstacle to the clinical application of klotho in the treatment of cancer.

According to one aspect, the present invention provides a method for inhibiting tumor growth in a subject in need thereof, the method comprising administering to said subject a gene transfer vector comprising a nucleotide sequence encoding a klotho protein operably linked to at least one regulatory sequence directing its expression.

In some embodiments, the klotho protein is a human klotho protein.

In some embodiments, the klotho protein is a soluble klotho protein selected from the group consisting of secreted klotho (s-KL), KL1 domain and KL1-KL2 fragment.

In some embodiments, the klotho protein is secreted klotho (s-KL) as set forth in SEQ ID NO: 1.

In some embodiments, the at least one regulatory sequence comprises a promoter selected from CMV, RSV and Desmin promoters.

In some particular embodiments, the klotho protein is secreted klotho (s-KL) and the at least one regulatory sequence comprises a CMV promoter. In additional particular embodiments, the klotho protein is secreted klotho (s-KL) and the at least one regulatory sequence comprises an RSV promoter.

In yet additional particular embodiments, the klotho protein is secreted klotho (s-KL) and the at least one regulatory sequence comprises a Desmin promoter.

An exemplary nucleotide sequence encoding s-KL operably linked to a CMV promoter is set forth at positions 175 to 2515 of SEQ ID NO: 2.

An exemplary nucleotide sequence encoding s-KL operably linked to an RSV promoter is set forth as SEQ ID NO: 3.

An exemplary nucleotide sequence encoding s-KL operably linked to a Desmin promoter is as set forth as SEQ ID NO: 4.

In some embodiments, the gene transfer vector is a viral vector.

In some embodiments, the gene transfer vector is a viral vector and the klotho protein is secreted klotho (s-KL) as set forth in SEQ ID NO: 1.

In some embodiments, the viral vector is an adeno-associated virus (AAV) viral vector.

In some embodiments, the viral vector is an AAV viral vector and the klotho protein is secreted klotho (s-KL) as set forth in SEQ ID NO: 1.

In some embodiments, the AAV viral vector is selected from AAV-5 and AAV-9.

In some embodiments, the AAV viral vector contains therein a nucleic acid sequence as set forth in SEQ ID NO: 2, comprising a sequence encoding secreted klotho (s-KL) operably linked to a CMV promoter, flanked by AAV Inverted Terminal Repeats (ITRs).

In some embodiments, the administering is systemically administering.

In some particular embodiments, systemically administering is administering via intramuscular injection.

In additional particular embodiments, systemically administering is administering via intravenous administration.

In some embodiments, the gene transfer vector is administered once.

In other embodiments, the gene transfer vector is administered at least twice.

In some embodiments, the subject is having a cancer selected from the group consisting of pancreatic, breast, colon, prostate, lung, cervical and ovarian cancer.

In some particular embodiments, the subject is having pancreatic cancer. These and further aspects and features of the present invention will become apparent from the detailed description, examples and claims which follow.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. a-klotho: membrane-bound klotho, soluble shed klotho, KL1, KL2 and differentially spliced secreted klotho prevalent in the brain (s-KL).

Figure 2. (A) pUC57-KL plasmid map; (B) Constructing AAV-sKL vector.

Figure 3. GFP expression levels in muscles of the nude mice obtained using AAV vectors of serotypes 1 , 2, 6, 8, 9 and 10.

Figure 4. Efficacy study overview.

Figure 5. Tumor volume reduction with low and high doses of AAV9-sKL gene therapy administered intramuscularly. +SEM is presented *=p<0.05.

Figure 6. Imaging of tumor size reduction with high dose AAV9-sKL treatment. (A) Qualitative images; (B) Quantitative analysis. Analysis of Mia Paca LUC-expressing cells in vivo. LUC signal is shown. -i-Std is presented **=p<0.005 in the quantification of LUC signal via count per minute. The images are sequential BLIs of the same animal.

Figure 7. Imaging of tumor size reduction with low dose AAV9-sKL treatment.

(A) Qualitative images; (B) Quantitative analysis. Analysis of Mia Paca LUC-expressing cells in vivo. LUC signal is shown. The images are sequential BLIs of the same animal.

Figure 8. Quantitative analysis of tumor size based on luciferase signal imaging. Quantification of LUC signal via count per minutes (cpm) (A) or photo per second (ph/s)

(B). -i-Std is presented *=p<0.05, **=p<0.005.

Figure 9. Quantitative analysis based on tumor weight. (A) Images of excised tumors; (B) Quantitative analysis. -i-Std is presented **=p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pharmaceutical compositions and treatment methods utilizing the same comprising gene therapy vectors carrying a nucleic acid sequence encoding a klotho protein. More particularly, the present invention relates to recombinant viral vectors, such as recombinant adeno-associated virus (rAAV) viral vectors, carrying a nucleic acid sequence encoding a klotho protein. The nucleic acid sequence encoding the klotho protein is under the control of regulatory sequences which direct expression of the klotho protein in cells of a subject in need of treatment. According to some embodiments, the treatment is directed to cancers such as pancreatic, breast and colon cancer.

To date, the most evident strategies to increase klotho levels in vivo include up-regulation of klotho using small molecules and administration of recombinant klotho proteins. However, up to date, small molecules have not been able to significantly and continuously increase klotho levels in the blood, and administration of recombinant klotho proteins cannot assure continuous high levels in the blood, and also it is unclear if recombinant klotho proteins can be produced consistently, at quantities and qualities required for clinical applications.

It was surprisingly found by the inventors of the present invention that significant tumor inhibition is obtained using the vectors disclosed herein. Without wishing to be bound by any particular theory or a mechanism to action, the significant tumor inhibition could be attributed to persistent high levels of klotho in the blood, such that the vectors enable robust production of klotho in vivo, at clinically-relevant amounts and quality.

The terms "nucleic acid sequence", "nucleotide sequence" and "polynucleotide" are used herein to refer to polymers of nucleotides, typically deoxyribonucleotides (DNA) and modified forms thereof, in the form of a separate fragment or as a component of a larger construct. A nucleic acid sequence may be a coding sequence, i.e., a sequence that encodes for an end product in the cell, such as a protein. A nucleic acid sequence may also be a regulatory sequence, such as, for example, a promoter.

The term "vector" refers to a man-made composition of matter which comprises a polynucleotide and which is designed for, and can be used to, deliver the polynucleotide to the interior of a target cell.

The term "expression", as used herein, refers to the cellular process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation.

The term "regulatory sequences" refer to DNA sequences which control the expression (transcription) of coding sequences. For example, regulatory sequences include promoters.

The term "promoter" is directed to a regulatory DNA sequence which drives or directs the transcription of another DNA sequence in vivo or in vitro. Usually, the promoter is located in the 5' region (that is, precedes, located upstream) of the transcribed sequence. Promoters may be derived in their entirety from a native source or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. Promoters can be constitutive (i.e. promoter activation is not regulated by an inducing agent and hence rate of transcription is constant), or inducible (i.e., promoter activation is regulated by an inducing agent). Promoters may be ubiquitous or cell/tissue-specific. In most cases the exact boundaries of regulatory sequences have not been completely defined, and in some cases cannot be completely defined, and thus DNA sequences of some variation may have identical promoter activity.

The terms "operably linked" and“under the control of’ mean that a selected nucleic acid sequence is located with respect to a regulatory sequence (e.g., a promoter) to allow the regulatory sequence to regulate expression of the selected nucleic acid sequence.

A "signal peptide" or "signal sequence" refers herein to a short peptide (usually 5- 30 amino acids long) typically present at the N-terminus of a newly synthesized polypeptide chain that directs the protein to the secretory pathway in the cell. The signal peptide is typically subsequently removed.

Viral vectors

Viral vectors for use according to the present invention are recombinant viral vectors. The viral vectors comprise a capsid with a polynucleotide construct packaged therein. The polynucleotide comprises a nucleic acid sequence encoding a klotho protein. The polynucleotide further comprises at least one regulatory sequence operably linked to the nucleic acid sequence encoding the klotho protein and directing its expression.

The at least one regulatory sequence typically comprises a promoter. Additional regulatory sequences include, for example, a polyadenylation signal, a terminator and the like.

Promoters suitable for use according to the present invention include ubiquitous promoters that are capable of driving/directing transcription in a constitutive matter in a wide range of cells, particularly in mammalian (preferably human) cells, as well as cell/tissue-specific promoters, such as muscle-specific or liver-specific promoters. Exemplary ubiquitous promoters suitable for use according to the present invention include a Cytomegalovirus (CMV) promoter and Rous sarcoma virus (RSV) promoter. In some particular embodiments, the CMV promoter is CMV-immediate early (CMV-IE) promoter. An exemplary CMV-IE promoter sequence is set forth at positions 175 to 860 of SEQ ID NO: 2. An exemplary RSV promoter sequence is set forth as SEQ ID NO: 5. An exemplary cell/tissue- specific promoter is Desmin promoter, which is muscle- specific. An exemplary Desmin promoter sequence is set forth as SEQ ID NO: 6.

An exemplary polynucleotide comprising a nucleic acid sequence encoding a klotho protein (s-KL) operably linked to a CMV-IE promoter, and further comprising a polyadenylation signal, is set forth at positions 175 to 2864 of SEQ ID NO: 2.

An exemplary nucleotide sequence encoding s-KL operably linked to a Desmin promoter is set forth as SEQ ID NO: 4.

In some embodiments, the at least one regulatory sequence comprises a promoter selected from CMV, RSV and Desmin promoters, and a polyadenylation signal.

An exemplary polyadenylation sequence is set froth at positions 2527 to 2864 of SEQ ID NO: 2.

The polynucleotide in the recombinant viral vector typically further comprises sequences that enable its packaging within the capsid. For example, the polynucleotide typically further comprises inverted terminal repeat (ITR) sequences.

In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. According to these embodiments, the viral vector comprises an AAV capsid. In addition, according to these embodiments, the polynucleotide in the capsid comprises AAV ITRs.

In some particular embodiments, the viral vector is an AAV9 viral vector. In additional embodiments, the viral vector is an AAV5 viral vector.

As used herein, the serotype refers to the capsid, for example,“AAV9 viral vector” indicates an AAV viral vector which contains the capsid of AAV serotype 9.

An exemplary polynucleotide to be included in a viral vector according to the present invention is set forth as SEQ ID NO: 2. The 5’-ITR corresponds to positions 1 to 145 of SEQ ID NO: 2. The CMV promoter corresponds to positions 175 to 860 of SEQ ID NO: 2. The nucleic acid sequence encoding the klotho protein (secreted klotho s-KL) corresponds to positions 861 to 2515 of SEQ ID NO: 2. The polyA sequence corresponds to positions 2527 to 2864 of SEQ ID NO: 2. The 3’-ITR corresponds to positions 2865 to 3008 of SEQ ID NO: 2.

AAV has a linear single- stranded DNA (ssDNA) genome of approximately 4.7-kilobases (kb), with two 145 nucleotide-long inverted terminal repeats (ITR) at the termini. The virus does not encode a polymerase and therefore relies on cellular polymerases for genome replication. The ITRs flank two viral genes - rep (replication) and cap (capsid), encoding non-structural and structural proteins, respectively. The rep gene, through the use of two promoters and alternative splicing, encodes four regulatory proteins, Rep78, Rep68, Rep52 and Rep40, which are involved in AAV genome replication. The cap gene, through alternative splicing and initiation of translation, gives rise to three capsid proteins, VP1, VP2 and VP3.

When AAV infects a human cell alone, its gene expression program is auto- repressed and latency is ensued by preferential integration of the virus genome into a specific region of roughly 2-kb on the long arm of human chromosome 19. This site- specific integration involves the AAV ITRs and Rep proteins. When a latently infected cell is co-infected with a helper virus, such as adenovirus or herpes simplex virus, the AAV gene expression program is activated leading to the AAV Rep-mediated rescue (i.e., excision) of the provirus DNA from the host cell chromosome, followed by replication and packaging of the viral genome.

In recombinant AAV vectors (rAAV), the r/is-acting viral DNA elements involved in genome amplification and packaging (namely, the ITRs) are typically in linkage with a heterologous sequence of interest (a transgene), whereas the region(s) encoding trans acting viral factors involved in genome replication and virion assembly (namely, the viral rep and cap genes) are not included and provided in trans.

Recombinant AAV vectors may be generated by transfecting producer cells with a plasmid (AAV r/.v-plasmid) containing a cloned recombinant AAV genome composed of the transgene flanked by the AAV ITRs, and a separate construct expressing in trans the viral rep and cap genes. Helper factors necessary for packaging of the transgene into the capsid are typically provided by transfecting into the producer cells a third plasmid that provides the helper factors. Alternatively, any one or more of the required components (e.g., rep sequences, cap sequences, and/or helper functions) may be provided by a stable producer cell which has been engineered to contain one or more of the required components. An exemplary procedure for generating rAAV vectors is described in more detail in the Examples section below. An exemplary sequence of a plasmid encoding rep and cap genes for AAV serotype 9 is set forth as SEQ ID NO: 7. An exemplary sequence of a plasmid encoding rep and cap genes for AAV serotype 5 is set forth as SEQ ID NO: 8. The resulting rAAV vector contains a polynucleotide with the transgene flanked by ITRs, packaged inside the capsid. Such rAAV vectors are generally incapable of integrating into the genome of their target cells, as they lack the Rep function. Such rAAV vectors are thought to persist inside the target cells’ nucleus as concatemers, providing long-term expression of the transgene.

The AAV ITRs, and other selected AAV components described herein, may be readily selected from among any AAV serotype, including, for example, AAV1 , AAV2, AAV3, AAV5, AAV6, AAV7, AAV8 and AAV9. Each possibility represents a separate embodiment of the present invention.

These ITRs or other AAV components may be readily isolated using techniques available to those of skill in the art from an AAV serotype. Such AAV may be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, Va.). Alternatively, the AAV sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like.

In some embodiments, the viral vectors of the present invention are AAV vectors that contain a nucleic acid sequence encoding a klotho protein and regulatory sequences, flanked by AAV 5' ITR and AAV 3' ITR, packaged inside an AAV capsid.

In some embodiments, the present invention utilizes an adeno-associated virus (AAV)-viral vector that comprises:

(a) a nucleic acid construct comprising a nucleic acid sequence encoding a klotho protein operably linked to at least one regulatory sequence, and AAV inverted terminal repeats (ITRs); and

(b) AAV capsid.

In some particular embodiments, the AAV capsid is AAV9 capsid. In additional particular embodiments, the AAV capsid is AAV5 capsid. In yet additional particular embodiments, the AAV ITRs are AAV2 ITRs.

Klotho proteins

In mice and humans, a -klotho gene encodes a transcript of ~5.2 kb, which contains five exons and four introns (see Figure 1). In the third exon, there is an alternative splicing donor site that can generate two different transcripts: one encoding a transmembrane form (full-length klotho, 1014 amino acids), and the other encoding a shorter, secreted form of the protein (550 amino acids). The transmembrane form, termed “membranal klotho” or

“m-KL”, is a single pass transmembrane protein with a molecular weight of approximately 135 kDa. It contains an N-terminal signal sequence, an extracellular domain with two internal repeats (KL1 and KL2, each is about 550 amino acids in length), a single transmembrane domain and a short intracellular domain (see Figure 1). The secreted form, termed“s-KL”, contains only the signal sequence and the KL1 domain, with a tail of additional 15 amino acids at the C-terminus. It has a molecular weight of approximately 70 kDa. The secreted form is abundant mainly in the brain.

The extracellular domain of the transmembrane form can be cleaved by metalloproteinases ADAM10 and ADAM17, resulting in another form of soluble klotho of about 130 kDa, termed“p-KL” for proteolyzed isoform. The p-KL isoform, composed of the KL1 and KL2 domains, has been detected in the serum, urine, and cerebrospinal fluid. A second recognition site for the proteases ADAM10 and ADAM17 is located between the KL1 and KL2 domains, resulting in two additional soluble forms, one is composed of the KL1 domain and the other is composed of the KL2 domain.

As used herein, the term“klotho protein” encompasses membranal klotho, secreted klotho, proteolyzed klotho (also referred to herein as“KL1-KL2” fragment), KL1 domain and KL2 domain. Each possibility represents a separate embodiment of the present invention. Preferably, the klotho protein is a human klotho protein.

The full amino acid sequence of human klotho is set forth as SEQ ID NO: 9.

The KL1 domain corresponds to positions 57 to 506 of SEQ ID NO: 9.

The KL2 domain corresponds to positions 517 to 953 of SEQ ID NO: 9.

Thus, the KL1-KL2 fragment corresponds to positions 57 to 953 of SEQ ID NO: 9.

The amino acid sequence of human secreted klotho (s-KL) is set forth as SEQ ID

NO: 1.

In some embodiments, the klotho protein for use according to the present invention is a soluble klotho protein, selected from: secreted klotho (s-KL), KL1-KL2 fragment and KL1 domain. In some particular embodiments, the klotho protein is secreted klotho (s- KL).

In some embodiments, the gene transfer vector for use according to the present invention is a gene transfer vector comprising a nucleotide sequence encoding s-KL operably linked to at least one regulatory sequence directing its expression. In some embodiments, the gene transfer vector is a viral vector comprising a nucleotide sequence encoding s-KL operably linked to at least one regulatory sequence directing its expression. In some embodiments, the gene transfer vector is an adeno-associated virus (AAV) viral vector comprising a nucleotide sequence encoding s-KL operably linked to at least one regulatory sequence directing its expression. In some particular embodiments, the gene transfer vector is an AAV-9 vector comprising a nucleotide sequence encoding s-KL operably linked to at least one regulatory sequence directing its expression.

In the context of the gene transfer vectors of the present invention, a nucleic acid encoding KL1 domain encompasses a nucleic acid encoding KL1 with a signal sequence that enables its secretion. In some embodiments, the KL1 domain corresponds to amino acids 1 -506 of SEQ ID NO: 9, which includes the natural signal sequence of klotho and the KL1 domain. Similarly, a nucleic acid encoding KL1-KL2 fragment encompasses nucleic acid encoding KL1-KL2 with a signal sequence that enables its secretion. In some embodiments, the KL1-KL2 fragment corresponds to amino acids 1-953 of SEQ ID NO: 9.

Nucleic acid sequences encoding the klotho proteins can be obtained using any of the many recombinant methods known in the art, for example by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned. A gene of interest may be cloned with a suitable signal sequence to enable its secretion.

As noted above, a nucleic acid sequence encoding a klotho protein according to the present invention is operably linked to at least one regulatory sequence. An exemplary nucleotide sequence encoding s-KL operably linked to at least one regulatory sequence comprising a CMV promoter and a polyadenylation signal is as set forth at positions 175 to 2868 of SEQ ID NO: 2.

The present invention further encompasses homologs of the nucleic acid sequences or the amino acid sequences of the present invention. A sequence (e.g., a nucleic acid sequence or an amino acid sequence) that is "homologous" to a reference sequence refers herein to percent identity between the sequences, where the percent identity is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99%. Each possibility represents a separate embodiment of the present invention. Sequence identity may be determined using nucleotide/amino acid sequence comparison algorithms, as known in the art.

Pharmaceutical compositions and uses

In some embodiments, there is provided herein a method for treating cancer in a subject in need thereof. More particularly, there is provided herein a method for inhibiting the growth of malignant tumors in subjects in need thereof. In additional embodiments, there is provided herein a pharmaceutical composition for use in the treatment of cancer. More particularly, there is provided herein a pharmaceutical composition for use in inhibiting the growth of malignant tumors in subjects in need thereof. The methods and compositions utilize vectors, particularly viral vectors, comprising a nucleotide sequence encoding a klotho protein operably linked to at least one regulatory sequence directing its expression, as described herein.

The subject to be treated according to the present invention is a mammalian subject, typically a human subject.

As used herein,“inhibition” of tumor growth encompasses attenuating, suppressing and even completely arresting the growth of malignant tumors. In some embodiments, the term also encompasses reducing tumor size.“Tumor size” as used herein refers to tumor volume, tumor weight or both. Reduction of tumor size may be complete, namely, destruction of the tumor.

In some embodiment, there is provided herein a method for reducing tumor size. In additional embodiments, there is provided herein a pharmaceutical composition for use in reducing tumor size.

In some embodiments, there is provided herein a method for inhibiting tumor growth in a subject with a solid tumor selected from a carcinoma, sarcoma and lymphoma. In some embodiments, the cancer is selected from the group consisting of pancreatic, breast, colon, prostate, lung, cervical and ovarian cancer.

In some additional embodiments, there is provided herein a method for inhibiting tumor growth in a subject with a cancer selected from pancreatic, breast and colon cancer.

In some embodiments, the cancer is an IGF- 1 -dependent cancer. In some embodiments, the cancer is selected from the group consisting of pancreatic, breast, colon, prostate, lung, cervical and ovarian cancer. In some particular embodiments, the cancer is pancreatic cancer (e.g., pancreatic ductal adenocarcinoma). In additional particular embodiments, the cancer is breast cancer. In yet additional particular embodiments, the cancer is colon cancer. In yet additional particular embodiments, the cancer is prostate cancer. In yet additional particular embodiments, the cancer is lung cancer. In yet additional particular embodiments, the cancer is cervical cancer. In yet additional particular embodiments, the cancer is ovarian cancer.

In some embodiments, the methods comprise systemically administering a viral vector expressing a klotho protein. Systemic administration according the present invention is parenteral systemic administration.

In some particular embodiments, the methods comprise administering via intramuscular injection a viral vector expressing a klotho protein. In some embodiments, the methods comprise administering via intramuscular injection an adeno-associated virus (AAV) viral vector, particularly AAV serotype 9, expressing a klotho protein.

In additional particular embodiments, the methods comprise administering via intravenous injection a viral vector expressing a klotho protein.

In some embodiments, the methods comprise administering via intrathecal administration a viral vector expressing a klotho protein.

In some embodiments, the viral vector is administered once. As provided herein, according to some embodiments, a single administration of the viral vector confers lasting expression of the klotho protein. For example, a single administration of the viral vector to a human subject may confer lasting expression for years, e.g., 1 -5, 10, 15 years, and possibly more.

In other embodiments, the viral vector is administered at least twice.

The recombinant viral vectors are typically formulated into a pharmaceutical composition suitable for systemic delivery, e.g., intramuscular or intravenous. Such formulation involves the use of a pharmaceutically acceptable vehicle or carrier, and optionally stabilizing agents, buffers, adjuvants, diluents, etc. For injection, the carrier is typically a liquid. Exemplary physiologically acceptable carriers include sterile water and sterile phosphate buffered saline.

In some embodiments, the recombinant viral vectors are formulated into a pharmaceutical composition suitable for intrathecal administration, which further comprises a suitable pharmaceutically acceptable carrier. In some embodiments, there is provided herein a method for inhibiting tumor growth in a subject in need, the method comprising administering to the subject an adeno- associated virus (AAV)-viral vector that comprises:

(b) AAV capsid,

wherein said administering results in increased levels of the klotho protein in the blood of the subject.

In some embodiments, a range for therapeutically effective amounts of a nucleic acid, nucleic acid construct, viral vectors or pharmaceutical composition may be from lxl0¹² and lxlO¹³ genome copy (gc)/kg, for example from lxlOⁿ to lxl0¹² gc/kg. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.

For gene therapy vectors, the dosage to be administered may depend to a large extent on the condition and weight of the subject being treated as well as the therapeutic formulation, frequency of treatment and the route of administration.

In some embodiments, the compositions and methods of the present invention result in increased levels of klotho in the blood compared to the levels in the blood prior to treatment. In some embodiments, the compositions and methods of the present invention result in inhibition of tumor growth. In some embodiments, the compositions and methods of the present invention result in reduction in tumor size.

List of sequences

The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Example 1 -AAV vectors expressing secreted klotho in a xenograft mouse model of pancreatic cancer

The following study tested the efficacy of administering an AAV vector expressing the secreted klotho isoform (“s-KL”) in a subcutaneous xenograft model of pancreatic cancer in mice. First, several AAV serotypes were examined in nude mice in order to select a serotype for expressing the klotho protein in intramuscular administration of the AAV vector. Next, the effect of administering an AAV-sKL vector was tested in a xenograft mouse model of pancreatic cancer.

Materials and methods

Construction of a plasmid vector encoding secreted klotho (s-KL)

A gene construct containing the cDNA of human s-KL, which is composed of the signal sequence, KL1 domain and a 15-amino acid tail, was generated from plasmid pUC57-KL (GenScript, USA). The plasmid map is shown schematically in Figure 2A.

The s-KL sequence was extracted from pUC57-KL using XbaI/Bspl20I restriction enzymes, and the resulting fragment was cloned in pGG2 (curtesy of Genethon), which contains a multiple cloning site between the Inverted Terminal Repeat (ITR) sequences of the adeno-associated virus serotype 2 (AAV2) (see Figure 2B). The plasmid pGG2 further contained a CMV-IE promoter. XbaI/Bspl20I that were used to cut pUC57-KL are compatible with Notl/Xbal, which were used to open the pGG2 plasmid. The resulting plasmid, containing the expression cassette CMV IE-sKL (with a polyadenylation signal) flanked with the ITRs, was termed“pGG2-sKL”.

The sequence of pGG2-sKL is set forth as SEQ ID NO: 2 and also provided below:

ITR-5’: lowercase, italics

Irrelevant sequence (for cloning purposes): lowercase

CMV promoter: lowercase, underlined sKL: uppercase

Irrelevant sequence (for cloning purposes): lowercase

PolyA sequence: lowercase, boldface

ITR-3’: lowercase, italics

PGG2-SKL:

aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgcccggg caaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagg gflgiggccflflctccatcactaggggttccttgtagttaa

tgattaacccgccatgctacttatctacgtagccatgctctagttattaatagtaactcaattacggggtcattag ttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacg acccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtca atgggtggactatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccct attgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttg gcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggat agcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaat caacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtg ggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacg actcactatagggagacccaagctggCT AGAATGCCCGCCAGCGCCCCGCCGC GCCGCCCGCGTCCACCGCCGCCGTCGCTGTCGCTGCTGCTGGTG CTGCTGGGCCTGGGCGGACGACGCCTGCGTGCGGAGCCGGGCG ACGGCGCGCAGACCTGGGCCCGTGTCTCGCGGCCTCCTGCCCCC GAGGCCGCGGGCCT CTTCC AGGGC ACCTT CCCCGACGGCTT CCT CTGGGCCGTGGGCAGCGCCGCCTACCAGACCGAGGGCGGCTGG CAGCAGCACGGCAAGGGTGCGTCCATCTGGGACACGTTCACCCA CCACCCCCTGGCACCCCCGGGAGACTCCCGGAACGCCAGTCTGC CGTTGGGCGCCCCGTCGCCGCTGCAGCCCGCCACCGGGGACGTA GCCAGCGACAGCTACAACAACGTCTTCCGCGACACGGAGGCGCT GCGCGAGCTCGGGGTCACTCACTACCGCTTCTCCATCTCGTGGG CGCGAGTGCTCCCCAATGGCAGCGCGGGCGTCCCCAACCGCGAG GGGCTGCGCTACTACCGGCGCCTGCTGGAGCGGCTGCGGGAGCT GGGCGTGCAGCCCGTGGTCACCCTGTACCACTGGGACCTGCCCC AGCGCCTGCAGGACGCCTACGGCGGCTGGGCCAACCGCGCCCTG GCCGACCACTTCAGGGATTACGCGGAGCTCTGCTTCCGCCACTT CGGCGGTC AGGT C A AGT ACTGGATC ACC AT CGAC A ACCCCT ACG TGGTGGCCTGGCACGGCTACGCCACCGGGCGCCTGGCCCCCGGC ATCCGGGGCAGCCCGCGGCTCGGGTACCTGGTGGCGCACAACCT CCTCCTGGCTCATGCCAAAGTCTGGCATCTCTACAATACTTCTTT CCGTCCCACTCAGGGAGGTCAGGTGTCCATTGCCCTAAGCTCTC ACTGGATCAATCCTCGAAGAATGACCGACCACAGCATCAAAGA ATGTCAAAAATCTCTGGACTTTGTACTAGGTTGGTTTGCCAAACC

CGTATTTATTGATGGTGACTATCCCGAGAGCATGAAGAATAACC

TTTCATCTATTCTGCCTGATTTTACTGAATCTGAGAAAAAGTTCA

TCAAAGGAACTGCTGACTTTTTTGCTCTTTGCTTTGGACCCACCT

T GAGTTTT C AACTTTTGGACCCT C AC ATGA AGTT CCGCC A ATTGG

AATCTCCCAACCTGAGGCAACTGCTTTCCTGGATTGACCTTGAAT

TTAACCATCCTCAAATATTTATTGTGGAAAATGGCTGGTTTGTCT

CAGGGACCACCAAGAGAGATGATGCCAAATATATGTATTACCTC

A A A A AGTT CAT CAT GGA A ACCTT A A A AGCC AT C A AGCTGGAT GG

GGTGGATGTCATCGGGTATACCGCATGGTCCCTCATGGATGGTT

T CG AGT GGC AC AG AGGTT AC AGC AT C AGGCGT GG ACT CTTCT AT

GTTGACTTTCTAAGCCAGGACAAGATGTTGTTGCCAAAGTCTTC

AGCCTTGTTCTACCAAAAGCTGATAGAGAAAAATGGCTTCCCTC

CTTTACCTGAAAATCAGCCCCTAGAAGGGACATTTCCCTGTGAC

TTT GCTT GGGG AGTT GT CG AC A ACT AC ATTC A AGT A AGT C AGCT

GACAAAACCAATCAGCAGTCTCACCAAGCCCTATCACTAGtaagcg

gccgc

ttccctttagtgagggttaatgcttcgagcagacatgataagatacattgatgagtttggacaaacca

caactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccatt

ataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggagat

gtgggaggttttttaaagcaagtaaaacctctacaaatgtggtaaaatccgataagggactagagca

tggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagzzg

gccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccggg

ctttgcccgggcggcctcagagagcgagcgagcgcgcagagagggagtggccaactccatcactag

ggttcct

It is noted that in this particular example plasmid pGG2 was used for cloning the s-KL sequence under the control of CMV IE promoter (and a polyadenylation signal), but other plasmids may be used, as long as they can be packaged in AAV capsids.

Control plasmids

Control GFP-expressing plasmids were provided by the Vector Production Unit at UAB Barcelona.

Production of AAV vectors for transduction

Recombinant AAV vectors containing pGG2-sKL were produced by triple transfection of 293-AAV cells from Stratagene (70% confluency) with: (i) the pGG2 plasmid (pGG2-sKL); (ii) pXX6 (courtesy of Genethon), an AAV helper plasmid; and (iii) pRep2Cap9 plasmid (SEQ ID NO: 7), carrying the AAV2 rep and the AAV9 cap genes. Following transfection viruses were purified using ultra centrifugation in iodixanol gradient, counted using PicoGreen® (see Piedra et al., 2015, Hum Gene Ther Methods, 26(1): 35-42), and stored at -80 °C until use. The viral vectors were named“AAV9-sKL”.

In particular, the production procedure was carried out as follows:

293-AAV cells (70% confluency in l5-cm diameter plates, total 20 plates) were transfected using polyethyleneimine (PEI) (PolyScience) with 500pg of pXX6, 250pg of pRep2Cap9 and 250pg of pGG2-sKL. The three plasmids were mixed in DMEM medium and added to the plates at a final volume of 14 ml/plate. 6 hours later the medium was changed. 48 hours post-transfection cells were scrapped and centrifuged. The cells were then resuspended in a lysis buffer (50 mM Tris (Sigma), 20 mM NaCl (Panreac) and 2 mM MgCF (Panreac)).

For AAV purification and after 3 cycles of frost and defrost, cell debris were separated by centrifugation and the supernatant was collected. Benzonase^® (50 U/ml) was added to the supernatant to degrade cellular DNA. Virus particles were further precipitated with polyethylene glycol (PEG, at 1 ml/4ml of cell lysate). Centrifugation at 8000g for 15 minutes allowed a pellet with the virus particles. l 5ml of the lysis buffer were then added to iodixanol gradient tubes and the virus particles removed after centrifugation at 690000 g for 1 hour.

AAV-GFP vectors were produced using the same procedure.

Selection of AAV serotype

AAV vectors of serotypes 1 , 2, 6, 8, 9 and 10 expressing GFP were produced as described above. Ad5-CMV vectors expressing GFP (Ad5-CMV-GFP) were used as positive control. The vectors were injected in one shot into the right flank muscle of female nude mice, 1.0E+l lgc/ml per injection per mouse. Three weeks later the mice were sacrificed and muscles were dissected and divided into four pieces. Each piece was homogenized (gentleMACS™, Miltenyi Biotec), RNA was extracted (Trizol) and RT- PCR was carried out (in triplicates) in order to evaluate the GFP expression levels obtained using each of the tested AAV serotypes. GFP was normalized to mouse b-actin.

GFP PCR primer: R-T C AGGT AGT GGTT GT CGGGC A (SEQ ID NO: 10)

GFP PCR primer: F-AAGCAGAAGAACGGCATCAAGGTG (SEQ ID NO: 11) b-actin primer: F- TGTT ACCAACTGGGACGAC A (SEQ ID NO: 12) b-actin primer: R- GGGGTGTT GAAGGTCTC AAA (SEQ ID NO: 13) Xenografts

lxlO⁶ mCherry/LUC labeled human Mia Paca-2 cells were suspended in IOOmI ECM gel (Sigma, cat no E1270) and injected subcutaneously to the right flank in each mouse.

Dose

High dose treatment: 5xl0^u virus particles (AAV9-sKL) per mouse.

Low dose treatment: 5xl0¹⁰ virus particles (AAV9-sKL) per mouse.

Control: 5x10¹¹ virus particles (AAV9-Null, expressing an untranslated sequence of the gene for the Homo sapiens transmembrane protein 185A (TMEM185A)) per mouse.

Administration

Each viral vector was administered once, intramuscularly, at the doses specified above. Time of treatment

Ten (10) days after injection of the cancer cells.

Termination of study

Animals were sacrificed two weeks after administration of the viral vectors. Timing determined based on tumor size.

Results

In mice and humans, a-klotho gene encodes a transcript of ~5.2 kb, which contains five exons and four introns (see Figure 1). In the third exon, there is an alternative splicing donor site that can generate two different transcripts: one encoding a transmembrane form (full-length klotho, 1014 amino acids), and the other encoding a shorter, secreted form of the protein (550 amino acids). The transmembrane form, termed “membranal klotho” or

“m-KL”, is a single pass transmembrane protein with a molecular weight of approximately 135 kDa. It contains an N-terminal signal sequence, an extracellular domain with two internal repeats (KL1 and KL2, each is about 550 amino acids in length), a single transmembrane domain and a short intracellular domain (see Figure 1). The amino acid sequence of the human membranal klotho is set forth as SEQ ID NO: 9 and provided below:

KL1 domain: boldface

KL2 domain: underline

Transmembrane domain: italics, boldface

Human membranal klotho:

MPASAPPRRPRPPPQSLSLLLVLLGLGGRRLRAEPGDGAQTWARFS RPPAPE AAGLFQGTFPDGFLWAV GSAAY QTEGGW QQHGKGASI WDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVF RDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRL LERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFR DYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGS PRLGYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWI NPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLS SILPDFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLES PNLRQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYL KKFIMETLKAIKLDGVDVIGYTAW SLMDGFEWHRGY SIRRGLF Y VDFLSODKMLLPKS SALFY OKLIEKN GFPPLPEN OPLEGTFPCD FAWGVVDNYIOVDTTLSOFTDLNVYLWDVHHSKRLIKVDGVVTK KRKSYCVDFAAIOPOIALLOEMHVTHFRFSLDWALILPLGNOSOVN HTILO Y YRCM ASELVR VNITP V VALW PPM APN OGLPRLL AROGA WENP YT ALAF AE Y ARLCF OELGHH VKL WITMNEP YTRNMT Y SAG HNLLKAHALAWHVYNEKFRHAONGKISIALOADWIEPACPFSOKD KEVAERVFEFDIGWFAEPIFGSGDYPWVMRDWFNORNNFFFPYFT EDEKKLIOGTFDFLALSHYTTILVDSEKEDPIKYNDYLEVOEMTDIT WLNSPSOVAVVPWGLRKVLNWLKFKYGDLPMYIISNGIDDGLHAE DDOLRVYYMONYINEALKAHILDGINLCGYFAYSFNDRTAPRFGL YRYAADOFEPKASMKHYRKIIDSNGFPGPETLERFCPEEFTVCTECS FFmRKSLLAFIAFLFFASIISLSLIFYYSKKGRRSYK

The secreted form, termed“s-KL”, contains only the protein sequence up to the KL1 domain (including) with a tail of additional 15 a ino acids at the C-terminus. It has a molecular weight of approximately 70 kDa. The secreted form is abundant mainly in the brain.

The current study tested the efficacy of an AAV vector expressing the secreted form of klotho, containing the KL1 domain with a tail of additional 15 amino acids at the C-terminus.

Selection of AAV serotype

As detailed above, AAV vectors of serotypes 1 , 2, 6, 8, 9, 10, and Ad5-CMV expressing GFP were produced and injected into muscles of mice. GFP expression levels in muscles of the mice obtained using each serotype are summarized in Figure 3. Particularly high expression was obtained using AAV9 and therefore this serotype was selected for expressing s-KL.

Efficacy of AAV-sKL

The study design is illustrated in Figure 4. Female nude mice (n=2l), 8 weeks old, were injected subcutaneously with lxlO⁶ mCherry/LUC labeled human Mia Paca-2 cells suspended in IOOmI ECM gel. Ten (10) days following tumor inoculation the mice were divided into three groups and injected intramuscularly, adjacent to the tumor, with either: AAV9-sKL high dose (n=7), AAV9-sKL low dose (n=6), or AAV9-Null (n=8) as a control.

Dimensions of the tumor were measured every two days and volume was calculated using the following formula: width x width x length / 2. Figure 5 summarizes the results, clearly demonstrating the beneficial effect of the treatment (*=p<0.05).

Further demonstration of the significant reduction in tumor volume in mice treated with the AAV-sKL vector is shown in Figures 6-8. The figures show analysis of the intensity of the luciferase signal from the Mia Paca-2 LUC-expressing cells in vivo, in mice injected with AAV-sKL high dose (Figure 6) or AAV-sKL low dose (Figure 7) compared to AAV-Null. Figure 8 shows quantitative analysis based on the luciferase signal imaging.

After study completion, tumors were excised, weighted and the results were compared between the three groups. As can be seen in Figure 9, tumor weight was significantly lower in the mice that were treated with AAV-sKL compared to mice that were treated with the control vector. To conclude, treatment with the AAV-sKL vector led to tumors with significantly reduced size at the end of the study compared to treatment with the control vector, indicating that growth of the tumors was significantly inhibited in mice that received the AAV-sKL vector. It is noted that the vectors were administered ten days following tumor inoculation, when tumors were already established in the mice. In addition, following administration, the active protein was not available immediately, but only after the time required for transfection of target cells and transcription and translation of the klotho protein, further strengthening the effect seen with the AAV-sKL vector in treating tumors and suppressing their growth.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed chemical structures and functions may take a variety of alternative forms without departing from the invention.

Claims

1. A method for inhibiting tumor growth in a subject in need thereof, the method comprising administering to said subject a gene transfer vector comprising a nucleotide sequence encoding a klotho protein operably linked to at least one regulatory sequence directing its expression, thereby inhibiting tumor growth in the subject.

2. The method of claim 1 , wherein said klotho protein is a human klotho protein.

3. The method of claim 1, wherein said klotho protein is a soluble klotho protein selected from the group consisting of secreted klotho (s-KL), KL1 domain and KL1-KL2 fragment.

4. The method of claim 1 , wherein said klotho protein is secreted klotho (s-KL) as set forth in SEQ ID NO: 1.

5. The method of claim 1, wherein said at least one regulatory sequence comprises a promoter selected from CMV, RSV and Desmin promoters.

6. The method of claim 1, wherein said klotho protein is secreted klotho (s-KL) and said at least one regulatory sequence comprises a CMV promoter.

7. The method of claim 1, wherein said klotho protein is secreted klotho (s-KL) and said at least one regulatory sequence comprises an RSV promoter.

8. The method of claim 1, wherein said klotho protein is secreted klotho (s-KL) and said at least one regulatory sequence comprises a Desmin promoter.

9. The method of claim 6, wherein the nucleotide sequence encoding s-KL operably linked to a CMV promoter is as set forth at positions 175 to 2515 of SEQ ID NO: 2.

10. The method of claim 7, wherein the nucleotide sequence encoding s-KL operably linked to an RSV promoter is as set forth in SEQ ID NO: 3.

11. The method of claim 8, wherein the nucleotide sequence encoding s-KL operably linked to a Desmin promoter is as set forth in SEQ ID NO: 4.

12. The method of claim 1 , wherein the gene transfer vector is a viral vector.

13. The method of claim 1 , wherein the gene transfer vector is a viral vector and wherein the klotho protein is secreted klotho (s-KL) as set forth in SEQ ID NO: 1.

14. The method of claim 12, wherein said viral vector is an adeno-associated virus (AAV) viral vector.

15. The method of claim 12, wherein said viral vector is an AAV viral vector and wherein the klotho protein is secreted klotho (s-KL) as set forth in SEQ ID NO: 1.

16. The method of claim 14, wherein the AAV viral vector is selected from AAV-5 and AAV-9.

17. The method of claim 16, wherein the AAV viral vector contains therein a nucleic acid sequence as set forth in SEQ ID NO: 2, comprising a sequence encoding secreted klotho (s-KL) operably linked to a CMV promoter, flanked by AAV Inverted Terminal Repeats (ITRs).

18. The method of claim 1 , wherein said administering is systemically administering.

19. The method of claim 18, wherein said systemically administering is administering via intramuscular injection.

20. The method of claim 18, wherein said systemically administering is administering via intravenous administration.

21. The method of claim 1 , wherein the gene transfer vector is administered once.

22. The method of claim 1 , wherein the gene transfer vector is administered at least twice.

23. The method of claim 1, wherein said subject is having a cancer selected from the group consisting of pancreatic, breast, colon, prostate, lung, cervical and ovarian cancer.

24. The method of claim 23, wherein said cancer is pancreatic cancer.