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US-12624076-B2 - AAV-EPO for treating companion animals

US12624076B2US 12624076 B2US12624076 B2US 12624076B2US-12624076-B2

Abstract

Compositions and methods are provided for treating companion animals are provided. An adeno-associated viral vector is provided which includes a nucleic acid molecule comprising a sequence encoding erythropoietin (EPO). In desired embodiments, the subject is a cat or dog.

Inventors

  • Christian Hinderer
  • James M. Wilson
  • Matthew Wilson

Assignees

  • THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA

Dates

Publication Date
20260512
Application Date
20210910

Claims (8)

  1. 1 . A method for treating chronic kidney disease in a feline or a canine subject, said method comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant adeno-associated virus (rAAV) comprising an AAV8 capsid having packaged therein a vector genome, wherein said vector genome comprises a nucleotide sequence encoding feline or canine erythropoietin (EPO), inverted terminal repeat sequences, and expression control sequences that direct expression of the EPO in a host cell, wherein the nucleotide sequence encoding the feline EPO comprises SEQ ID NO: 8, and wherein the nucleotide sequence encoding the canine EPO comprises SEQ ID NO: 7.
  2. 2 . The method according to claim 1 , wherein the encoded feline EPO comprises the amino acids 27 to 192 of SEQ ID NO: 4 in combination with a heterologous leader sequence.
  3. 3 . The method according to claim 1 , wherein the feline EPO nucleotide sequence encodes amino acids 1 to 192 of SEQ ID NO: 4.
  4. 4 . The method according to claim 1 , wherein the encoded canine EPO amino acid sequence comprises the amino acids 41 to 206 of SEQ ID NO: 3 in combination with a heterologous leader sequence.
  5. 5 . The method according to claim 1 , wherein the canine EPO nucleotide sequence encodes amino acids 1 to 206 of SEQ ID NO: 3.
  6. 6 . The method according to claim 1 , wherein the expression control sequences comprise a promoter.
  7. 7 . The method according to claim 6 , wherein the promoter is selected from a CB7 promoter, a TBG promoter, a Nkcc2 promoter, a uromodulin promoter, a Ksp-cadherin promoter, and a THP gene promoter.
  8. 8 . The method according to claim 1 , wherein the expression control sequences comprise one or more of an intron, a Kozak sequence, a polyA, and post-transcriptional regulatory elements.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 15/754,931, filed Feb. 23, 2018, which application is a 371 National Stage Entry of International Patent Application No. PCT/US2016/049487, filed Aug. 30, 2016, which application claims the benefit of the priority of U.S. Provisional Patent Application No. 62/336,211, filed May 13, 2016, and U.S. Provisional Patent Application No. 62/212,144, filed Aug. 31, 2015. Each of these applications are incorporated herein by reference. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM Applicant hereby incorporates by reference the Sequence Listing material filed in electronic form herewith. This file is labeled “15-7472PCT_Seq_Listing.txt”. BACKGROUND OF THE INVENTION Erythropoietin (EPO) is a hormone made predominantly within the peritubular cells of the kidney. It acts on the bone marrow, stimulating erythropoiesis. Erythropoietin also controls apoptosis (programmed cell death) of mature red blood cells. Renal disease reduces erythropoietin production. In humans, the management of anemia in chronic kidney disease has been revolutionized by the development of recombinant human erythropoietin (epoetin). Many of the symptoms that had been ascribed to chronic kidney disease such as fatigue, lethargy, somnolence and shortness of breath, which all impact unfavorably on quality of life, were resolved or markedly improved when anemia was corrected. There are over 2 million cats and 350,000 dogs that suffer from chronic kidney disease (CKD). Companion animals with CKD-related renal failure suffer in similar ways. They do not have sufficient EPO and subsequently become very anemic. In the past veterinarians have given human recombinant EPO until the animals would develop an immune response to the infused EPO. Effectively, this leaves no long term treatment in the market for a very well understood physiological process that has a clear need in the clinic. Therefore, compositions useful for expressing EPO in subjects, particularly companion animals, are needed. SUMMARY OF THE INVENTION Novel engineered erythropoietin (EPO) constructs are provided herein. These constructs can be delivered to subjects in need thereof via a number of routes, and particularly by expression in vivo mediated by a recombinant vector such as a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the EPO is encoded by an endogenous sequence. That is, the EPO sequence is derived from the same subject species for which administration is ultimately intended. In some embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant vector as described herein is provided. Also provided are methods for treating chronic kidney disease by administering to a subject in need thereof a recombinant vector described herein that has an expression cassette, wherein said expression cassette further comprises regulatory control sequences which direct expression of the EPO construct in the subject. In some embodiments, the subject being treated is a companion animal. In one embodiment, the subject is a feline. In another embodiment, the subject is a canine. As used herein, the terms “patient” and “subject” are used interchangeably, and can refer to a human or veterinary subject. In yet another embodiment, methods for increasing the amount of circulating EPO in a subject comprising providing a recombinant vector described herein that has an expression cassette encoding EPO. The recombinant vectors described above can be used in a regimen for treating chronic kidney disease and other conditions characterized by a decrease in the amount of circulating red blood cells. Other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing hematocrits of cats treated with AAV8 expressing feline erythropoietin. Dashed lines indicate the normal range. FIG. 2 is a graph showing hematocrits of dogs treated with AAV8 expressing canine erythropoietin. Dashed lines indicate the normal range. FIG. 3A shows the canine EPO propeptide sequence, with the leader sequence underlined. FIG. 3B shows the feline EPO propeptide sequence, with the leader sequence underlined. FIG. 4 is a graph showing hematocrits of cats treated with AAV8 expressing feline erythropoietin. Cats were treated with 3.0×107 GC, 3.0×108 GC, 3.0×109 GC, or 3.0×1010 GC AAV8f.EPO. DETAILED DESCRIPTION OF THE INVENTION Adeno-associated viral vectors carrying EPO expression constructs have been developed for use in subjects including companion animals (e.g., feline and canine). Though likely effective, a recombinant canine or feline specific EPO protein therapeutic would cost much more to develop and manufacture than a viral vector mediated system for delivery of EPO to the affected animal. With a viral vector therapeut