In vivo gene therapy of hemophilia B: sustained partial correction in factor IX-deficient dogs

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.

Download Full-text

Neonatal or hepatocyte growth factor–potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B

Blood ◽

10.1182/blood-2002-10-3050 ◽

2003 ◽

Vol 101 (10) ◽

pp. 3924-3932 ◽

Cited By ~ 73

Author(s):

Lingfei Xu ◽

Cuihua Gao ◽

Mark S. Sands ◽

Shi-Rong Cai ◽

Timothy C. Nichols ◽

...

Keyword(s):

Gene Therapy ◽

Growth Factor ◽

Gene Transfer ◽

Hepatocyte Growth Factor ◽

Retroviral Vector ◽

Factor Ix ◽

Hemophilia B ◽

Hepatocyte Growth

AbstractHemophilia B is a bleeding disorder resulting from factor IX (FIX) deficiency that might be treated with gene therapy. Neonatal delivery would correct the disease sooner than would transfer into adults, and could reduce immunological responses. Neonatal mice were injected intravenously with a Moloney murine leukemia virus–based retroviral vector (RV) expressing canine FIX (cFIX). They achieved 150% to 280% of normal cFIX antigen levels in plasma (100% is 5 μg/mL), which was functional in vitro and in vivo. Three newborn hemophilia B dogs that were injected intravenously with RV achieved 12% to 36% of normal cFIX antigen levels, which improved coagulation tests. Only one mild bleed has occurred during 14 total months of evaluation. This is the first demonstration of prolonged expression after neonatal gene therapy for hemophilia B in mice or dogs. Most animals failed to make antibodies to cFIX, demonstrating that neonatal gene transfer may induce tolerance. Although hepatocytes from newborns replicate, those from adults do not. Adult mice therefore received hepatocyte growth factor to induce hepatocyte replication prior to intravenous injection of RV. This resulted in expression of 35% of normal cFIX antigen levels for 11 months, although all mice produced anti-cFIX antibodies. This is the first demonstration that high levels of FIX activity can be achieved with an RV in adults without a partial hepatectomy to induce hepatocyte replication. We conclude that RV-mediated hepatic gene therapy is effective for treating hemophilia B in mice and dogs, although the immune system may complicate gene transfer in adults.

Download Full-text

In vivo hepatic gene therapy: complete albeit transient correction of factor IX deficiency in hemophilia B dogs.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.6.2353 ◽

1994 ◽

Vol 91 (6) ◽

pp. 2353-2357 ◽

Cited By ~ 178

Author(s):

M. A. Kay ◽

C. N. Landen ◽

S. R. Rothenberg ◽

L. A. Taylor ◽

F. Leland ◽

...

Keyword(s):

Gene Therapy ◽

Factor Ix ◽

Hemophilia B ◽

Factor Ix Deficiency

Download Full-text

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 ◽

10.1182/blood.v110.11.5136.5136 ◽

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.

Download Full-text

Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

10.1101/2021.03.18.435908 ◽

2021 ◽

Author(s):

Xi Chen ◽

Xuran Niu ◽

Yang Liu ◽

Rui Zheng ◽

Liren Wang ◽

...

Keyword(s):

Gene Therapy ◽

Specific Activity ◽

High Specific Activity ◽

Factor Ix ◽

Hemophilia B ◽

Hereditary Diseases ◽

Proliferating Cells ◽

Promising Therapeutic Approach ◽

Targeted Integration

Site-specific integration of exogenous gene through genome editing is a promising strategy for gene therapy. However, homology-directed repair (HDR) only occurring in proliferating cells is inefficient especially in vivo. To investigate the efficacy of Cas9-induced homology-independent targeted integration (HITI) strategy for gene therapy, a rat hemophilia B model was generated and employed. Through HITI, a DNA sequence encoding the last exon of rat Albumin (rAlb) gene fused with a high-specific-activity Factor IX variant (R338L) using T2A, was inserted into the last intron of rAlb via recombinant adeno-associated viral (rAAV). The knock-in efficiency reached up to 3.66% determined by ddPCR. The clotting time was reduced to normal level 4 weeks after treatment, and the circulating FIX level was gradually increased up to 52% of normal during 9 months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through PEM-seq, no significant off-targeting effect was detected. Moreover, this study provides a promising therapeutic approach for hereditary diseases.

Download Full-text

Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

10.21203/rs.3.rs-302699/v1 ◽

2021 ◽

Author(s):

Dali Li ◽

Xi Chen ◽

Xuran Niu ◽

Yang Liu ◽

Rui Zheng ◽

...

Keyword(s):

Gene Therapy ◽

Specific Activity ◽

High Specific Activity ◽

Factor Ix ◽

Hemophilia B ◽

Hereditary Diseases ◽

Proliferating Cells ◽

Promising Therapeutic Approach ◽

Targeted Integration

Abstract Site-specific integration of exogenous gene through genome editing is a promising strategy for gene therapy. However, homology-directed repair (HDR) only occurring in proliferating cells is inefficient especially in vivo. To investigate the efficacy of Cas9-induced homology-independent targeted integration (HITI) strategy for gene therapy, a rat hemophilia B model was generated and employed. Through HITI, a DNA sequence encoding the last exon of rat Albumin (rAlb) gene fused with a high-specific-activity Factor IX variant (R338L) using T2A, was inserted into the last intron of rAlb via recombinant adeno-associated viral (rAAV). The knock-in efficiency reached up to 3.66% determined by ddPCR. The clotting time was reduced to normal level 4 weeks after treatment, and the circulating FIX level was gradually increased up to 52% of normal during 9 months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through PEM-seq, no significant off-targeting effect was detected. Moreover, this study provides a promising therapeutic approach for hereditary diseases.

Download Full-text

Extravascular Administration of Factor IX: Potential for Replacement Therapy of Canine and Human Hemophilia B

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656082 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0944-0948 ◽

Cited By ~ 20

Author(s):

Darla Liles ◽

Charles N Landen ◽

Dougald M Monroe ◽

Celeste M Lindley ◽

Marjorie s Read ◽

...

Keyword(s):

Gene Therapy ◽

Vitamin K ◽

Absorption Rate ◽

Venous Access ◽

Factor Ix ◽

Hemophilia B ◽

Current Therapy ◽

Home Therapy ◽

Routes Of Administration ◽

Plasma Compartment

SummaryCurrent therapy for hemophilia B requires large intravenous doses of factor IX (F.IX) given in the clinic or at home. Although home therapy is possible for many patients, it is often complicated by factors such as the lack of good venous access. Very little is known about extravascular routes for administering proteins like F.IX (57 kD) or other vitamin K-dependent procoagulant factors into the circulation. Questions about the absorption rate from extravascular administration as well as plasma recovery and bioavailability have arisen recently with the growing availibility of highly purified procoagulant proteins and increased interest in gene therapy of hemophilia B. Therefore, a group of studies were undertaken to determine the absorption rate, plasma recovery, and bioavailability of high purity, human plasma-derived F.IX concentrates administered via extravascular routes in hemophilia B dogs and in one human hemophilia B subject. Five hemophilia B dogs were given human F.IX via either a subcutaneous (SC), intramuscular (IM), intra- peritoneal (IP) or intravenous (IV) route. In a subsequent study, a single SC administration of human F.IX was compared to an identical IV dose of F.IX in the human hemophilia B subject. All extravascular routes of F.IX administration in both the canine and human gave lower levels of circulating plasma F.IX than the IV route, however all routes resulted in measurable F.IX activity. Of the extravascular routes, the IM injection in the canine resulted in a bioavailibility of 82.8%, while the SC injection resulted in a bioavailability of 63.5%. F.IX reached the plasma compartment by all extravascular routes used, confirming that F.IX can be absorbed extravascularly. The duration of measurable F.IX activity following extravascular administration is prolonged beyond that typically seen with IV administration. These data show that significant levels of F.IX may be obtained via SC injection in canine and ‘ human hemophilia B subjects and further highlight the potential of extravascular routes of administration for future experimental and clinical uses of F.IX and other procoagulant proteins.

Download Full-text

Faculty Opinions recommendation of Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732234317.793540850 ◽

2017 ◽

Author(s):

Bruno Stieger

Keyword(s):

Gene Therapy ◽

Specific Activity ◽

High Specific Activity ◽

Factor Ix ◽

Hemophilia B ◽

Activity Factor

Download Full-text

Factor IX variants improve gene therapy efficacy for hemophilia B

Blood ◽

10.1182/blood-2004-08-2990 ◽

2005 ◽

Vol 105 (6) ◽

pp. 2316-2323 ◽

Cited By ~ 45

Author(s):

Joerg Schuettrumpf ◽

Roland W. Herzog ◽

Alexander Schlachterman ◽

Antje Kaufhold ◽

Darrel W. Stafford ◽

...

Keyword(s):

Skeletal Muscle ◽

Intramuscular Injection ◽

Specific Activity ◽

Factor Ix ◽

Hemophilia B ◽

Promising Strategy ◽

B Mice ◽

Higher Doses ◽

Vector Delivery

Abstract Intramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.

Download Full-text