scholarly journals Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver

Blood ◽  
2006 ◽  
Vol 107 (7) ◽  
pp. 2653-2661 ◽  
Author(s):  
Amit C. Nathwani ◽  
John T. Gray ◽  
Catherine Y. C. Ng ◽  
Junfang Zhou ◽  
Yunyu Spence ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Human Factor ◽  
Factor Ix ◽  
Expression Cassette ◽  
Hemophilia B ◽  
Adeno Associated Virus ◽  
Knock Out ◽  
Human Factor Ix ◽  
Murine Liver ◽  
Knock Out Mice

AbstractTransduction with recombinant adeno-associated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAV-mediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini–human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles. This unique property resulted in a 20-fold improvement in hFIX expression in mice over comparable ssAAV vectors. Administration of only 1 × 1010 scAAV particles led to expression of hFIX at supraphysiologic levels (8I U/mL) and correction of the bleeding diathesis in FIX knock-out mice. Of importance, therapeutic levels of hFIX (3%-30% of normal) were achieved in nonhuman primates using a significantly lower dose of scAAV than required with ssAAV. Furthermore, AAV5-pseudotyped scAAV vectors mediated successful transduction in macaques with pre-existing immunity to AAV8. Hence, this novel vector represents an important advance for hemophilia B gene therapy.

A Novel Human Factor IX Expression Cassette Packaged as Self-Complementary DNA Dimers in Adeno-Associated Virus Vectors Results in Dramatically Improved Liver Transduction, Significantly Improving Prospects for Hemophilia B Gene Therapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3181-3181
Author(s):  
John T. Gray ◽  
Andrew M. Davidoff ◽  
Amit C. Nathwani
Keyword(s):  
Gene Therapy ◽  
Human Factor ◽  
Factor Ix ◽  
Expression Cassette ◽  
Adeno Associated Virus ◽  
Complementary Dna ◽  
Virus Vectors ◽  
Tail Vein ◽  
Human Factor Ix ◽  
Stable Plasma

Abstract Current approaches for gene therapy of hemophilia B (HB) are focused on liver targeted delivery of recombinant adeno-associated virus vectors (rAAV) encoding human Factor IX (hFIX). A major rate limiting step to efficient transduction of the liver is the need to convert the single stranded rAAV genome to a double stranded transcriptionally active form by the host cell. Recently described self-complementary AAV (scAAV) vectors, which can package AAV transgenes as DNA dimers when they are half the size of the wild type genome, bypass the need for second strand synthesis. Their substantially smaller packaging capacity has, however, limited their use for HB gene therapy. We have overcome this obstacle by designing a mini-human FIX expression cassette that is efficiently packaged as self-complementary DNA dimers in rAAV vectors. The key aspects of this novel expression cassette include a truncated regulatory element (LP1) consisting of the critical domains of human apolipoprotein E/C-I gene locus control region and the α1-antitrypsin (hAAT) promoter, and very small intron and polyadenylation elements from SV40. The hFIX 3′untranslated region has been deleted and the coding sequence was re-constructed (hFIXco) using a subset of codons most frequently found in highly expressed eukaryotic genes. The resulting expression cassette, which spanned 2.15 kbp, was cloned into an AAV-2 vector in which the right terminal resolution site had been deleted. Encapsidation with AAV-8 capsid proteins produced the scAAV-2/8 LP1-hFIXco vector in yields comparable to single stranded rAAV. Hirt analysis indicated that all vector genomes in murine liver after tail vein administration existed in a double stranded conformation. Stable plasma hFIX levels at 50% of physiologic (2867 ± 501 ng/ml) were achieved after tail vein administration of 2 x 109 scAAV-2/8 LP1-hFIXco vector/mouse. This is >500 fold higher than levels achieved with standard single stranded rAAV-2 vectors and >10 fold higher than levels achieved with a comparable single stranded vector pseudotyped with AAV-8 capsid proteins, confirming the superiority of scAAV vectors. Further increases in scAAV-2/8 LP1-hFIXco vector dose to 1 x 1011 particles/mouse resulted in a proportionately linear increase in stable plasma hFIX levels to over 100 μg/ml without any adverse toxicity. Importantly, transgene expression mediated by the LP1 promoter/enhancer appears to be strictly restricted to the liver. Evaluation of the efficacy and safety of scAAV-2/8 LP1-hFIXco in rhesus macaques is ongoing. However, based on the murine studies, our novel self complementary hFIX expressing vector offers a unique opportunity to mediate therapeutic gene transfer in humans without the need for higher vector doses. This has important safety implications and will therefore substantially improve the prospects of hemophilia gene therapy.

scholarly journals Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5095-5103 ◽  
Author(s):  
G Hortelano ◽  
A Al-Hendy ◽  
FA Ofosu ◽  
PL Chang
Keyword(s):  
Gene Therapy ◽  
Human Factor ◽  
Ex Vivo ◽  
Cost Effective ◽  
Factor Ix ◽  
Hemophilia B ◽  
Therapy Strategy ◽  
Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B

2015 ◽  
Vol 9 (1) ◽  
pp. 90-99 ◽  
Author(s):  
Ru Zhang ◽  
Qiang Wang ◽  
Lin Zhang ◽  
Saijuan Chen
Keyword(s):  
Gene Therapy ◽  
Human Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Human Factor Ix

scholarly journals Etranacogene Dezaparvovec (AAV5-Padua hFIX variant), an Enhanced Vector for Gene Transfer in Adults with Severe or Moderate-Severe Hemophilia B: Two Year Data from a Phase 2b Trial

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 13-13
Author(s):  
Annette von Drygalski ◽  
Adam Giermasz ◽  
Giancarlo Castaman ◽  
Nigel S. Key ◽  
Susan U. Lattimore ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Board Of Directors ◽  
Research Funding ◽  
Factor Ix ◽  
Hemophilia B ◽  
Advisory Committees ◽  
Adeno Associated Virus ◽  
Human Factor Ix ◽  
Virus Serotype ◽  
Serotype 5

Background: Gene therapy for hemophilia offers the possibility to ameliorate disease severity to a mild or functionally curative state through a single administration. Etranacogene dezaparvovec (AMT-061) is an investigational gene therapy for hemophilia B comprising an adeno associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) gene with liver specific promoter. Aims: We have previously shown a single dose of etranacogene dezaparvovec to provide sustained FIX activity into the mild-to normal range for up to 52 weeks post-dose in participants with severe or moderate-severe hemophilia B. This time, 2 years of follow-up data will be presented for the first time. Methods: A Phase 2b, open-label, single-dose, single-arm, multi-center trial (NCT03489291) in adult hemophilia B subjects. Interestingly, participants were not excluded based on neutralizing antibodies to AAV5. All subjects received a single intravenous dose of etranacogene dezaparvovec (2x1013 gc/kg) and will be followed for 5-years. The primary endpoint was FIX activity at Week 6. Secondary endpoints include e-diary recordings of bleeds and FIX concentrate use, laboratory parameters, joint health, patient reported outcomes, and adverse events (AEs). Results: All participants had FIX ≤1% (severe or moderately-severe FIX deficiency), required routine FIX prophylaxis, and had neutralizing activity to AAV5 at baseline. Following AMT-061 treatment, FIX activity increased rapidly to a mean of 31% at Week 6. At Week 52, mean FIX activity increased further to 41% with FIX activity levels of 50%, 31% and 41% in participants 1-3 respectively. There was no relationship between the presence of anti-AAV5 NAbs and response to etranacogene dezaparvovec. As of 52 weeks, there were no bleeds post-treatment and no requirement for FIX replacement aside from protocol-specified use for perioperative management in participant 3. There were no clinically significant elevations in liver enzymes and no participants required steroids related to the treatment. One participant experienced 2 mild AEs possibly related to treatment shortly after dosing (self-limiting headache and slightly elevated CRP). One patient had hip surgery due to worsening of pre-existing avascular necrosis deemed unrelated by investigator to etranacogene dezaparvovec and received FIX per protocol according to standard clinical practice. No participant developed inhibitors to FIX. Updated results to 2 years of follow-up will be presented with the main focus on FIX activity, FIX replacement therapy use and reported bleeds. Conclusions: Patients with AAV5 NAbs were included in the Phase 2b etranacogene dezaparvovec trial and have shown sustained FIX activity into the mild-to normal range. All participants were able to discontinue routine prophylaxis, and there have been no bleeds post-treatment with etranacogene dezaparvovec. Disclosures Giermasz: BioMarin: Consultancy, Research Funding, Speakers Bureau; Genentech/Roche: Consultancy, Research Funding, Speakers Bureau; uniQure: Consultancy, Research Funding; Sangamo Therapeutics: Research Funding; Bioverativ/Sanofi: Consultancy, Research Funding, Speakers Bureau. Castaman:Alexion: Honoraria; Roche: Consultancy, Honoraria, Speakers Bureau; CSL Behring: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ablynx: Honoraria; Baxalta/Shire: Honoraria; Bayer: Honoraria; Uniqure: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kedrion: Speakers Bureau; Werfen: Speakers Bureau; Sobi: Honoraria, Research Funding, Speakers Bureau; Novo Nordisk: Honoraria, Speakers Bureau. Key:Novo Nordisk: Other: Chair of Grants Committee; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy. Miesbach:Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BioMarin Pharmaceutical Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; UniQure: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Recht:Spark: Research Funding; Novo Nordisk: Consultancy, Other: personal fees, Research Funding; uniQure: Consultancy, Other: personal fees, Research Funding; Takeda: Consultancy, Other: personal fees, Research Funding; BioMarin: Research Funding; Pfizer: Consultancy, Other: personal fees; Genentech: Consultancy, Other: personal fees, Research Funding; CSL Behring: Consultancy, Other: personal fees. Gomez:Global Blood Therapeutics: Speakers Bureau. Gut:uniQure: Current Employment. Pipe:Siemens: Research Funding; Medical and Scientific Advisory Council to the National Hemophilia Foundation; Medical Advisory Board to World Federation of Hemophilia: Membership on an entity's Board of Directors or advisory committees; Apcintex, Bayer, BioMarin, Catalyst Biosciences, CSL Behring, HEMA Biologics, Freeline, Novo Nordisk, Pfizer, F. Hoffmann-La Roche Ltd/Genentech, Inc., Sangamo Therapeutics, Sanofi, Takeda, Spark Therapeutics, uniQure: Consultancy. OffLabel Disclosure: Etranacogene dezaparvovec (AMT-061) is an investigational gene therapy for hemophilia B comprising an adeno associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) gene with liver specific promoter.

Mesenchymal Stromal Cells Gene-Enhanced To Express Human Factor IX and Their In Vivo Use for Correction of Hemophilia B Phenotype in R333Q Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5136-5136
Author(s):  
Daniel L. Coutu ◽  
Jessica Cuerquis ◽  
May Griffith ◽  
Mark D. Blostein ◽  
Jacques Galipeau
Keyword(s):  
Gene Therapy ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Human Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Monogenic Disease ◽  
Human Factor Ix ◽  
B Mice

Abstract Hemophilia B is considered an appropriate disease target for gene therapy because it is a well characterized monogenic disease with a large therapeutic index. Despite promising preclinical and clinical trials in the last decade, safety and efficacy concerns associated with the in vivo administration of viral vectors still need to be addressed before gene therapy becomes part of the standard arsenal for clinicians. Our laboratory has developed a cell therapy approach using gene-enhanced autologous Mesenchymal Stromal Cells (MSCs) to deliver a therapeutic plasmatic protein which addresses these safety concerns. In this study, we tested whether MSCs engineered to express human Factor IX (hFIX) can be used to reverse the bleeding phenotype of R333Q hemophilia B mice developed by Stafford et al. We retrovirally engineered MSCs harvested from normal C57Bl/6 to express hFIX. A gene enhanced polyclonal population of MSCs was capable of producing carboxylated and fully active hFIX by in vitro clotting assays. By ELISA, the cells were shown to produce approximately 250ng of hFIX per million cells per 24h. Ten million of these cells were embedded in a collagen I gel matrix and implanted subcutaneously in R333Q hemophilia B mice (n=10). hFIX activity in mouse plasma (test and control groups) were followed weekly by aPTT assays. hFIX activity reached levels as high as 20% normal activity in some animals with an average +/− SEM of 11.2 +/− 2.1 (FIX activity in controls is <1%). The hFIX activity returned to baseline within 4 weeks. In conclusion, we demonstrate that gene-enhanced autologous MSCs can serve as an effective delivery of functional FIX for temporary correction of the hemophilia B phenotype. We hypothesize the presence of GFP co-expression by the implanted MSCs caused their immune rejection and we are currently testing this hypothesis.

scholarly journals Gene therapy with adeno-associated virus vector 5–human factor IX in adults with hemophilia B

Blood ◽  
2018 ◽  
Vol 131 (9) ◽  
pp. 1022-1031 ◽  
Author(s):  
Wolfgang Miesbach ◽  
Karina Meijer ◽  
Michiel Coppens ◽  
Peter Kampmann ◽  
Robert Klamroth ◽  
...  
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Cellular Immune Responses ◽  
Human Factor Ix ◽  
Key Points ◽  
Cellular Immune ◽  
Observation Period

Key Points AAV5 liver-directed wild-type hFIX gene transfer was well tolerated and clinically effective in severe and moderate-severe hemophilia B. No cellular immune responses to the AAV5 vector were detected, and FIX expression levels were stable for the entire observation period.

The Rhesus Macaque as an Animal Model for Hemophilia B Gene Therapy

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 1875-1881 ◽  
Author(s):  
Jay N. Lozier ◽  
Mark E. Metzger ◽  
Robert E. Donahue ◽  
Richard A. Morgan
Keyword(s):  
Gene Therapy ◽  
Nucleotide Sequence ◽  
Rhesus Macaque ◽  
Half Life ◽  
Messenger Rna ◽  
Human Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Human Cdna ◽  
Human Factor Ix

We have determined the 2905 nucleotide sequence of the rhesus macaque factor IX complementary DNA (cDNA) and found it to be greater than 95% identical to that of the human factor IX cDNA. The cDNA has a large 3′ untranslated region like the human cDNA, but unlike the human cDNA has two polyadenylation sites 224 nucleotides apart that are used for transcription of the messenger RNA. The deduced amino acid sequence is greater than 97% identical to that of human factor IX, differing in only 11 of 461 amino acids in the complete precursor protein. We found a single silent polymorphism in the nucleotide sequence at the third position of the codon for asparagine at position 167 in the secreted protein (AAC/AAT). All residues subject to posttranslational modifications in the human protein are also found in the rhesus factor IX sequence. The high degree of homology between the rhesus and human factor IX proteins suggested the possibility that the human factor IX protein might be nonimmunogenic in the rhesus. We tested the immunogenicity of human factor IX in three rhesus macaques by repeated intravenous injections of monoclonal antibody–purified, plasma-derived human factor IX over the course of more than a year and assessed the recovery and half-life of the infused protein, as well as in vitro indicators of antihuman factor IX antibodies. Human factor IX recovery and half-life remained unchanged over the course of a year in the three animals studied, and aPTT mixing studies showed no evidence for neutralizing antihuman factor IX antibodies. An outbred, nonhuman primate model that permits assessment of the level and duration of factor IX expression as well as vector safety would complement the use of other (mouse and canine) hemophilia B animal models in current use for the development of gene therapy for hemophilia B.

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