403. A Novel In-Vivo Approach for AAV-Mediated Gene Therapy of Hemophilia B Using Salivary Glands as a Depot Organ

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.

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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.

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In vivo enrichment of genetically manipulated platelets for murine hemophilia B gene therapy

Journal of Cellular Physiology ◽

10.1002/jcp.29861 ◽

2020 ◽

Vol 236 (1) ◽

pp. 354-365

Author(s):

Yingyu Chen ◽

Jocelyn A. Schroeder ◽

Chunyan Gao ◽

Jing Li ◽

Jianda Hu ◽

...

Keyword(s):

Gene Therapy ◽

Hemophilia B ◽

Genetically Manipulated

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Targeted Modifications in Adeno-Associated Virus Serotype (AAV)- 8 Capsid Improves Its Hepatic Gene Transfer Efficiency in Vivo

Blood ◽

10.1182/blood.v120.21.2045.2045 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2045-2045

Author(s):

Dwaipayan Sen ◽

Nishanth Gabriel ◽

Sathish Kumar Yesupatham ◽

Rekha Samuel ◽

Rupali A Gadkari ◽

...

Keyword(s):

Gene Therapy ◽

Conflicts Of Interest ◽

Innate Immune ◽

Hemophilia B ◽

Transduction Efficiency ◽

Egfp Expression ◽

Adeno Associated Virus ◽

Virus Serotype ◽

Antibody Levels

Abstract Abstract 2045 Recombinant adeno-associated virus vectors based on serotype (AAV)-8 have shown significant promise for liver directed gene therapy of hemophilia B. However, in a recent clinical trial, two patients who received highest dose (2×1012 vg/kg) of the self-complementary (sc)AAV8 vector developed capsid specific T cells that required glucocorticoid therapy to attenuate this response [Nathwani et al, New Eng J Med, 2011]. Thus, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery, we modified specific serine/threonine kinase or ubiquitination targets on AAV8 capsid to improve its transduction efficiency. To test this, point mutations at specific serine (S), threonine (T) or lysine (K) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant capsids were evaluated for their liver transduction efficiency at a dose of 5 × 1010 vgs/ animal in C57BL/6 mice in vivo. Two of the AAV8-S>A mutants (S279A and S501A) and a K137R mutant vector, demonstrated significantly higher EGFP expression (3.6 to 12.5 fold) in the liver compared to animals that received WT-AAV8 vectors alone (Figure 1). The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response [interleukin (IL)-6, IL-12, tumor necrosis factor α, Kupffer cells (KC) and innate immune responsive toll like receptors (TLR)-9] with a concomitant 2-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies also revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (22 fold), lungs (9.7 fold) and muscle (8.4 fold) tissue when compared to WT-AAV8 vectors. Further on-going studies with the optimal mutant scAAV8 vector expressing human coagulation factor IX in murine models of hemophilia B, will demonstrate the feasibility of the use of these novel vectors for potential gene therapy of hemophilia B. Figure 1: Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Figure 1:. Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Disclosures: No relevant conflicts of interest to declare.

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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

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Gene therapy in PIDs, hemoglobin, ocular, neurodegenerative, and hemophilia B disorders

Open Life Sciences ◽

10.1515/biol-2021-0033 ◽

2021 ◽

Vol 16 (1) ◽

pp. 431-441

Author(s):

Arome Solomon Odiba ◽

Nkwachukwu Oziamara Okoro ◽

Olanrewaju Ayodeji Durojaye ◽

Yanjun Wu

Keyword(s):

Gene Therapy ◽

Gene Transfer ◽

Globin Gene ◽

Severe Combined Immunodeficiency ◽

Retinal Disease ◽

Cell Types ◽

Biologically Active ◽

Hemophilia B ◽

Combined Immunodeficiency

Abstract A new approach is adopted to treat primary immunodeficiency disorders, such as the severe combined immunodeficiency (SCID; e.g., adenosine deaminase SCID [ADA-SCID] and IL-2 receptor X-linked severe combined immunodeficiency [SCID-X1]). The success, along with the feasibility of gene therapy, is undeniable when considering the benefits recorded for patients with different classes of diseases or disorders needing treatment, including SCID-X1 and ADA-SCID, within the last two decades. β-Thalassemia and sickle cell anemia are two prominent monogenic blood hemoglobin disorders for which a solution has been sought using gene therapy. For instance, transduced autologous CD34+ HSCs via a self-inactivating (SIN)-Lentivirus (LV) coding for a functional copy of the β-globin gene has become a feasible procedure. adeno-associated virus (AAV) vectors have found application in ocular gene transfer in retinal disease gene therapy (e.g., Leber’s congenital amaurosis type 2), where no prior treatment existed. In neurodegenerative disorders, successes are now reported for cases involving metachromatic leukodystrophy causing severe cognitive and motor damage. Gene therapy for hemophilia also remains a viable option because of the amount of cell types that are capable of synthesizing biologically active FVIII and FIX following gene transfer using AAV vectors in vivo to correct hemophilia B (FIX deficiency), and it is considered an ideal target, as proven in preclinical studies. Recently, the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 gene-editing tool has taken a center stage in gene therapy research and is reported to be efficient and highly precise. The application of gene therapy to these areas has pushed forward the therapeutic clinical application.

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Current therapeutic relevance of liver gene transfer Kay MA, Rothenberg S, Landen CN, Bellinger DA, Leland F, Toman C, Finegold M, Thompson AR, Read MS, Brinkhous KM, Woo SLC. In vivo gene therapy of hemophilia B: sustained partial correction on factor IX-deficient dogs. Science 1993;262:117?119

Hepatology ◽

10.1016/0270-9139(94)90161-9 ◽

1994 ◽

Vol 20 (1) ◽

pp. 253-254 ◽

Cited By ~ 1

Author(s):

J HEARD

Keyword(s):

Gene Therapy ◽

Gene Transfer ◽

Factor Ix ◽

Hemophilia B ◽

Therapeutic Relevance ◽

Liver Gene ◽

Partial Correction

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In vivo Endocrine Secretion of Prostacyclin Following Expression of a Cyclooxygenase-1/Prostacyclin Fusion Protein in the Salivary Glands of Rats Via Nonviral Gene Therapy

Human Gene Therapy ◽

10.1089/hum.2017.040 ◽

2017 ◽

Vol 28 (8) ◽

pp. 681-689 ◽

Cited By ~ 1

Author(s):

Zhimin Wang ◽

Raymond L. Benza ◽

Lee Zourelias ◽

Angela Sanguino ◽

Ramaz Geguchadze ◽

...

Keyword(s):

Gene Therapy ◽

Fusion Protein ◽

Salivary Glands ◽

Nonviral Gene Therapy ◽

Cyclooxygenase 1 ◽

Endocrine Secretion

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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.

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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.

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