OPTIMIZED AAV-MEDIATED HUMAN FACTOR VIII GENE THERAPY IN HEMOPHILIA A MICE AND CYNOMOLGUS MACAQUES
Author(s): ,
Jenny A Greig
Affiliations:
Gene Therapy Program, Department of Medicine,University of Pennsylvania,Philadelphia,United States
,
Qiang Wang
Affiliations:
Gene Therapy Program, Department of Medicine,University of Pennsylvania,Philadelphia,United States
,
Amanda L Reicherter
Affiliations:
Gene Therapy Program, Department of Medicine,University of Pennsylvania,Philadelphia,United States
,
Erin Bote
Affiliations:
Gene Therapy Program, Department of Medicine,University of Pennsylvania,Philadelphia,United States
,
Deirdre McMenamin
Affiliations:
Gene Therapy Program, Department of Medicine,University of Pennsylvania,Philadelphia,United States
,
Christine Draper
Affiliations:
Gene Therapy Program, Department of Medicine,University of Pennsylvania,Philadelphia,United States
,
Shu-Jen Chen
Affiliations:
Gene Therapy Program, Department of Medicine,University of Pennsylvania,Philadelphia,United States
,
Alexandra L Hanlon
Affiliations:
School of Nursing,University of Pennsylvania,Philadelphia,United States
,
Tamara Goode
Affiliations:
Gene Therapy Program, Department of Medicine,University of Pennsylvania,Philadelphia,United States
,
K Reed Clark
Affiliations:
Dimension Therapeutics,Cambridge,United States
,
Samuel Wadsworth
Affiliations:
Dimension Therapeutics,Cambridge,United States
,
Lili Wang
Affiliations:
Gene Therapy Program, Department of Medicine,University of Pennsylvania,Philadelphia,United States
James M Wilson
Affiliations:
Gene Therapy Program, Department of Medicine,University of Pennsylvania,Philadelphia,United States
(Abstract release date: 05/19/16) EHA Library. A Greig J. 06/10/16; 133353; P366
Dr. Jenny A Greig
Dr. Jenny A Greig
Contributions
Abstract
Abstract: P366

Type: Poster Presentation

Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45

Location: Poster area (Hall H)

Background
Hemophilia A is an X-linked bleeding disorder driven by a deficiency in human coagulation factor VIII (hFVIII), which occurs at a frequency of 1 in 5,000 live male births. The bleeding manifestations of hemophilia, which directly correlate with circulating FVIII activity, can result in significant disability or even death. Protein replacement therapy is effective in maintaining a mild or moderate phenotype. However, infusion frequency remains burdensome to patients. AAV-based gene therapy has the potential to provide long-term improvement of the disease phenotype following a single administration of vector.

Aims
In an effort to optimize expression of hFVIII for the treatment of hemophilia A, an extensive study was performed combining liver-specific promoter and enhancer elements with a codon-optimized human B-domain-deleted hFVIII transgene.

Methods
Due to the large size of the FVIII coding sequence, it is critical for gene expression control elements to be as short as possible while retaining hepatocyte-restricted transcription. Therefore, several strong liver-specific promoters were shortened and combined, with combinations consisting of up to three liver-specific enhancer sequences, to generate 42 enhancer/promoter combinations. These 42 liver regulatory gene cassettes were then packaged into the AAVrh10 capsid and tested in FVIII KO mice. Following intravenous (IV) administration of 1010 genome copies (GC), mice were bled every 2 weeks to follow hFVIII activity and antibody generation to the transgene.

Results
At week 2 post-injection, mice showed a range in hFVIII activity from 0.12-2.12 IU/ml. FVIII KO mice developed antibodies to hFVIII at week 4, and by week 8, mice in most of the 42 vector groups had detectable anti-hFVIII IgG levels. Based on the FVIII KO mouse studies and a small pilot rhesus macaque study, 2 of the original 42 enhancer/promoter combinations were selected for further evaluation in cynomolgus macaques, using 2 different Clade E capsids for expression. Each of the 4 vector combinations were administered IV at a dose of 1.2x1013 GC/kg into 5 macaques per group. With one capsid plus enhancer/promoter combination, peak expression of 37% of normal FVIII levels was seen at week 2 post-vector administration, which then plateaued at 20% of normal. While antibodies to the hFVIII were detected in the majority of macaques by week 8, antibodies remained undetectable in 2 animals at week 30 post-vector administration.

Conclusion
The use of an AAVrh10 vector to deliver a codon-optimized, B-domain-deleted hFVIII transgene results in a substantial increase in FVIII expression in both FVIII KO mice and cynomolgus macaques. The majority of animals generated an anti-FVIII antibody response by week 8 post vector delivery. However, 2 of the macaques dosed were free of a detectable antibody response through week 30. Overall, our study supports the continued development of AAV-based gene therapeutics for hemophilia A.

Session topic: Gene therapy, cellular immunotherapy and vaccination (Poster)

Keyword(s): AAV, Adeno-associated virus, Gene therapy, Hemophilia A
Abstract: P366

Type: Poster Presentation

Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45

Location: Poster area (Hall H)

Background
Hemophilia A is an X-linked bleeding disorder driven by a deficiency in human coagulation factor VIII (hFVIII), which occurs at a frequency of 1 in 5,000 live male births. The bleeding manifestations of hemophilia, which directly correlate with circulating FVIII activity, can result in significant disability or even death. Protein replacement therapy is effective in maintaining a mild or moderate phenotype. However, infusion frequency remains burdensome to patients. AAV-based gene therapy has the potential to provide long-term improvement of the disease phenotype following a single administration of vector.

Aims
In an effort to optimize expression of hFVIII for the treatment of hemophilia A, an extensive study was performed combining liver-specific promoter and enhancer elements with a codon-optimized human B-domain-deleted hFVIII transgene.

Methods
Due to the large size of the FVIII coding sequence, it is critical for gene expression control elements to be as short as possible while retaining hepatocyte-restricted transcription. Therefore, several strong liver-specific promoters were shortened and combined, with combinations consisting of up to three liver-specific enhancer sequences, to generate 42 enhancer/promoter combinations. These 42 liver regulatory gene cassettes were then packaged into the AAVrh10 capsid and tested in FVIII KO mice. Following intravenous (IV) administration of 1010 genome copies (GC), mice were bled every 2 weeks to follow hFVIII activity and antibody generation to the transgene.

Results
At week 2 post-injection, mice showed a range in hFVIII activity from 0.12-2.12 IU/ml. FVIII KO mice developed antibodies to hFVIII at week 4, and by week 8, mice in most of the 42 vector groups had detectable anti-hFVIII IgG levels. Based on the FVIII KO mouse studies and a small pilot rhesus macaque study, 2 of the original 42 enhancer/promoter combinations were selected for further evaluation in cynomolgus macaques, using 2 different Clade E capsids for expression. Each of the 4 vector combinations were administered IV at a dose of 1.2x1013 GC/kg into 5 macaques per group. With one capsid plus enhancer/promoter combination, peak expression of 37% of normal FVIII levels was seen at week 2 post-vector administration, which then plateaued at 20% of normal. While antibodies to the hFVIII were detected in the majority of macaques by week 8, antibodies remained undetectable in 2 animals at week 30 post-vector administration.

Conclusion
The use of an AAVrh10 vector to deliver a codon-optimized, B-domain-deleted hFVIII transgene results in a substantial increase in FVIII expression in both FVIII KO mice and cynomolgus macaques. The majority of animals generated an anti-FVIII antibody response by week 8 post vector delivery. However, 2 of the macaques dosed were free of a detectable antibody response through week 30. Overall, our study supports the continued development of AAV-based gene therapeutics for hemophilia A.

Session topic: Gene therapy, cellular immunotherapy and vaccination (Poster)

Keyword(s): AAV, Adeno-associated virus, Gene therapy, Hemophilia A

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