scholarly journals 34. Retroviral Vector-Mediated Neonatal Gene Therapy Results in Long-Term and Therapeutic Levels of Canine Factor VIII Expression in Hemophilia A Mice and Dogs

2004 ◽  
Vol 9 ◽  
pp. S14-S15
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Retroviral Vector

Animal Testing of Retroviral-Mediated Gene Therapy for Factor VIII Deficiency

1999 ◽  
Vol 82 (08) ◽  
pp. 555-561 ◽  
Author(s):  
Douglas Jolly ◽  
Judith Greengard
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Joint Damage ◽  
Coagulation Factor ◽  
The United States ◽  
Severe Hemophilia ◽  
Factor Viii Gene ◽  
Bleeding Episodes

IntroductionHemophilia A results from the plasma deficiency of factor VIII, a gene carried on the X chromosome. Bleeding results from a lack of coagulation factor VIII, a large and complex protein that circulates in complex with its carrier, von Willebrand factor (vWF).1 Severe hemophilia A (<1% of normal circulating levels) is associated with a high degree of mortality, due to spontaneous and trauma-induced, life-threatening and crippling bleeding episodes.2 Current treatment in the United States consists of infusion of plasma-derived or recombinant factor VIII in response to bleeding episodes.3 Such treatment fails to prevent cumulative joint damage, a major cause of hemophilia-associated morbidity.4 Availability of prophylactic treatment, which would reduce the number and severity of bleeding episodes and, consequently, would limit such joint damage, is limited by cost and the problems associated with repeated venous access. Other problems are associated with frequent replacement treatment, including the dangers of transmission of blood-borne infections derived from plasma used as a source of factor VIII or tissue culture or formulation components. These dangers are reduced, but not eliminated, by current manufacturing techniques. Furthermore, approximately 1 in 5 patients with severe hemophilia treated with recombinant or plasma-derived factor VIII develop inhibitory humoral immune responses. In some cases, new inhibitors have developed, apparently in response to unnatural modifications introduced during manufacture or purification.5 Gene therapy could circumvent most of these difficulties. In theory, a single injection of a vector encoding the factor VIII gene could provide constant plasma levels of factor in the long term. However, long-term expression after gene transfer of a systemically expressed protein in higher mammals has seldom been described. In some cases, a vector that appeared promising in a rodent model has not worked well in larger animals, for example, due to a massive immune response not seen in the rodent.6 An excellent review of early efforts at factor VIII gene therapy appeared in an earlier volume of this series.7 A summary of results from various in vivo experiments is shown in Table 1. This chapter will focus on results pertaining to studies using vectors based on murine retroviruses, including our own work.

Toward Gene Therapy for Hemophilia A: Long-Term Persistence of Factor VIII-Secreting Fibroblasts after Transplantation into Immunodeficient Mice

1993 ◽  
Vol 4 (2) ◽  
pp. 179-186 ◽  
Author(s):  
Rob C. Hoeben ◽  
Frits J. Fallaux ◽  
Nico H. Van Tilburg ◽  
Steve J. Cramer ◽  
Hans Van Ormondt ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Immunodeficient Mice

A Novel Deletion in the Fviii B-Domain That Reduces Transgene Size While Preserving FVIII Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3182-3182
Author(s):  
Yi-Lin Liu ◽  
Hua Zhu ◽  
Alexander Schlachterman ◽  
Heesoon Chang ◽  
Rodney M. Camire ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Dose Group ◽  
Hemophilia A ◽  
Specific Activity ◽  
Post Injection ◽  
Vector Genome ◽  
Fviii Activity

Abstract Hemophilia A is an inherited X-linked bleeding disorder caused by a deficiency in Factor VIII (FVIII). Clinically significant improvement of hemophilia phenotype can be achieved with low circulating factors, thus makes it a good target disease for gene therapy. Adeno-associated virus (AAV) vectors have proven successful for the delivery of the factor IX gene in humans with hemophilia B. For the treatment of hemophilia A, a problem in the packaging of the rFVIII cDNA or various B-domainless derivatives (i.e. rFVIII-SQ) in AAV vectors is the large size of the insert, which combined with required elements, can exceed the packaging capacity of AAV (~5 kb). This difficulty limits the choice of both promoter and regulatory elements when designing an expression cassette for AAV vectors. Here we developed strategies to overcome these limitations by (1) development of a novel FVIII B-domain deleted molecule (2) construction of a short liver-specific promoter. We further tested these vectors in a series of in vitro and in vivo experiments. Factor VIII-SQ is a well-characterized derivative of FVIII and has been used by several groups in a gene therapy setting; the recombinant protein is used clinically to treat hemophilia A. We have constructed a shorter version of FVIII-SQ, by deleting the entire B-domain. In addition, we have engineered this FVIII to be intracellularly processed using a PACE-furin recognition site such that the protein is secreted from cells as two chains (FVIII-RKR; fully processed heavy and light chains). This FVIII-RKR along with FVIII-SQ was transiently expressed in COS-1 cells and conditioned media was collected at 24, 48 and 72 hrs post transfection. Using a combination of ELISA and functional assays we were able to demonstrate that FVIII-RKR was efficiently secreted from these cells. The data also revealed that FVIII-RKR has a 4–8-fold increase in specific activity compared to FVIII-SQ. We further tested whether FVIII-RKR could function in an in vivo setting. Plasmid DNA (50μg) containing FVIII-RKR or FVIII-SQ with liver-specific mouse transthyretin (mTTR) promoter were introduced into hemophilia A (HA) mice hydrodynamically via tail vein. Two out of four mice in the SQ group and three out of four mice in the RKR group had significant shortening of the clotting time at days 1 and 3 post injection, indicating that this shortened version of FVIII is functional in vivo. To address FVIII long-term expression we synthesized AAV vectors and delivered to immuno-deficient HA mice through hepatic portal vein. AAV vectors containing an expression cassette of mTTR promoter and FVIII-SQ have been administered. Expression of physiological FVIII levels was observed in high dose group (4.0E+12 vector genome per animal, n=4). FVIII activity averages 1.88 U/ml by Coamatic assay or 0.81 U/ml by aPTT assay at 12 weeks post injection. In low dose group (1.0E+12 vector genome per animal, n=5) therapeutic level of FVIII is achieved, 0.59 U/ml by Coamatic assay or 0.23 U/ml by aPTT assay at 12 weeks post injection. Finally, AAV vectors with FVIII-RKR have been produced and shown to have similar packaging efficiency to AAV-FVIII-SQ. Studies are currently underway with AAV-FVIII-RKR to evaluate the ability of this vector to drive long-term expression of functional protein. In summary, we developed a novel FVIII molecule that has high specific activity and is suitable for efficiently packaging in the AAV vectors.

scholarly journals Long-Term AAV-Mediated Factor VIII Expression in Nine Hemophilia A Dogs: A 10 Year Follow-up Analysis on Durability, Safety and Vector Integration

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 611-611 ◽  
Author(s):  
Giang N. Nguyen ◽  
John K. Everett ◽  
Hayley Raymond ◽  
Samita Kafle ◽  
Elizabeth P. Merricks ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Liver Function ◽  
Factor Viii ◽  
Copy Number ◽  
Hemophilia A ◽  
Clonal Expansion ◽  
Clotting Factor ◽  
Single Chain

Hemophilia is an X-linked bleeding disorder caused by a deficiency in clotting factor VIII (FVIII)(hemophilia A, HA) or factor IX (FIX)(hemophilia B, HB). While early clinical trials of AAV delivery of FIX for HB have demonstrated stable FIX expression for &gt;8 years, an ongoing clinical trial of AAV-FVIII delivery for HA achieved high levels of transgene expression that unexpectedly declined after 1 year. Here we describe preclinical studies of AAV-canine FVIII (cFVIII) delivery in nine HA dogs with sustained FVIII expression for the duration of the study, as long as 10 years. FVIII was delivered using two delivery approaches: (1) co-administration of two AAV vectors encoding separate cFVIII heavy and light chains driven by the thyroxine binding globulin (TBG) promoter (Two chain approach)(TC) (n=5) at two AAV doses (2.5 x 1013vg/kg; F24, Woodstock, J60) and (1.2 x 1013vg/kg; Linus, H19) or (2) delivery of cFVIII as a single chain driven by the human alpha-1 anti-trypsin (hAAT) promoter (Single chain approach)(SC)(n=4) at two AAV doses (4 x 1013 vg/kg; M50, M06) and (2 x 1013vg/kg; M66, L51) (Sabatino 2011). We demonstrated that both strategies were efficacious; preventing &gt;95% of spontaneous bleeding episodes without toxicity. We now report the long-term follow-up of between 2.2 and 10.1 years for these treated dogs. Dose-dependent cFVIII:C (Coatest SP4 FVIII) was observed. At the final time point, the cFVIII:C was 2.7% (F24), 7.1% (Woodstock), 4.5% (J60), 11.3% (Linus) and 2.5% (H19) for TC dogs. For the SC dogs, the cFVIII:C was 9.4% (M06), 10.3% (M50), 1.9% (L51) and 3.7% (M66). Stable FVIII expression was maintained for seven of the dogs over the course of the study. Two dogs (Linus, M50) had a gradual increase in FVIII:C that began about three years after vector administration and continued for an additional seven years (Linus) and four years (M50), until the termination of the study. Liver function tests, serum alpha-fetoprotein concentrations, fibrinogen levels as well as liver pathology did not suggest altered liver function or tumor development in Linus and M50 compared to the other dogs. Clinically, there was no evidence of malignancy and no tumors were detected at the time of necropsy in any dog. One of the safety concerns for AAV-mediated gene therapy approaches is the potential for AAV integration events to be genotoxic and lead to tumorigenesis. While recombinant AAV primarily remains as an episome, integration events have been observed in mouse models and hepatocellular carcinoma has been observed after neonatal delivery of AAV vectors. In addition, the increase in FVIII expression in Linus and M50 prompted us to investigate integration and clonal expansion as a potential mechanism for these observations. Vector copy number (VCN) analysis was performed on liver samples (5-29 per dog, n=8 dogs) by Q-PCR and detected DNA copy numbers between 0.0 and 7.8 per diploid genome (Fig 1A). We performed integration target site analysis on liver samples (n=3/dog) from six of the AAV-treated HA dogs and naïve HA dogs (n=2) by ligation-mediated PCR, Ilumina paired-end sequencing and analysis using the custom software pipeline, AAVenger. Analysis of the 20 samples identified &gt;2,000 unique AAV integration events (IE). There was a correlation between the DNA copy number and the number of integration events detected. Clonal abundances were estimated by counting the unique genome breaks associated with integration positions, which showed that the maximum clonal abundance ranged from 1 to 138. The integration events were distributed across the canine genome. Clonal expansions were observed with integration near genes previously associated with growth control and transformation in humans (Fig 1B) with the most abundant clones located in DLEU2L (Linus), PEBP4 (J60) and EGR3 (M50). Integration events in EGR3, EGR2, CCND1, LTO1 and ZNF365 were detected in multiple dogs. Validation of integration sites in the most abundant clones was performed using targeted PCR to isolate junction fragments followed by Sanger sequencing. While AAV integration and clonal expansion was observed, the dogs had no evidence for tumorigenesis and it is not clear if the increase in FVIII expression is associated with the clonal expansions detected. Overall, these studies demonstrate long-term sustained FVIII expression for up to 10 years with clonal expansion, but without clinical adverse events after AAV-mediated gene therapy for hemophilia. Disclosures Sabatino: Spark Therapeutics: Patents & Royalties.

scholarly journals Anti-CD3 antibodies modulate anti–factor VIII immune responses in hemophilia A mice after factor VIII plasmid-mediated gene therapy

Blood ◽  
2009 ◽  
Vol 114 (20) ◽  
pp. 4373-4382 ◽  
Author(s):  
Baowei Peng ◽  
Peiqing Ye ◽  
David J. Rawlings ◽  
Hans D. Ochs ◽  
Carol H. Miao
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Growth Factor ◽  
Immune Responses ◽  
Factor Viii ◽  
Transforming Growth Factor ◽  
Hemophilia A ◽  
Transforming Growth Factor Β ◽  
Inhibitory Antibodies

Abstract One major obstacle in gene therapy is the generation of immune responses directed against transgene product. Five consecutive anti-CD3 treatments concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic levels of FVIII gene expression in treated hemophilia A mice. Repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+ and CD8+ T cells, whereas increased transforming growth factor-β levels in plasma and the frequency of both CD4+CD25+FoxP3+ and CD4+CD25−Foxp3+ regulatory T cells in the initial few weeks after treatment. Although prior depletion of CD4+CD25+ cells did not abrogate tolerance induction, adoptive transfer of CD4+ cells from tolerized mice at 6 weeks after treatment protected recipient mice from anti-FVIII immune responses. Anti-CD3–treated mice mounted immune responses against both T-dependent and T-independent neo-antigens, indicating that anti-CD3 did not hamper the immune systems in the long term. Concomitant FVIII plasmid + anti-CD3 treatment induced long-term tolerance specific to FVIII via a mechanism involving the increase in transforming growth factor-β levels and the generation of adaptive FVIII-specific CD4+Foxp3+ regulatory T cells at the periphery. Furthermore, anti-CD3 can reduce the titers of preexisting anti-FVIII inhibitory antibodies in hemophilia A mice.

scholarly journals Therapeutic factor VIII levels and negligible toxicity in mouse and dog models of hemophilia A following gene therapy with high-capacity adenoviral vectors

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1734-1743 ◽  
Author(s):  
Marinee K. L. Chuah ◽  
Gudrun Schiedner ◽  
Lieven Thorrez ◽  
Brian Brown ◽  
Marion Johnston ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
High Capacity ◽  
Scid Mice ◽  
Therapeutic Window ◽  
Expression Levels ◽  
Cellular Immune

High-capacity adenoviral (HC-Ad) vectors expressing B-domain–deleted human or canine factor VIII from different liver-specific promoters were evaluated for gene therapy of hemophilia A. Intravenous administration of these vectors into hemophilic FVIII-deficient immunodeficient SCID mice (FVIIIKO-SCID) at a dose of 5 × 109 infectious units (IU) resulted in efficient hepatic gene delivery and long-term expression of supraphysiologic FVIII levels (exceeding 15 000 mU/mL), correcting the bleeding diathesis. Injection of only 5 × 107 IU still resulted in therapeutic FVIII levels. In immunocompetent hemophilic FVIII-deficient mice (FVIIIKO), FVIII expression levels peaked at 75 000 mU/mL but declined thereafter because of neutralizing anti-FVIII antibodies and a cellular immune response. Vector administration did not result in thrombocytopenia, anemia, or elevation of the proinflammatory cytokine interleukin-6 (IL-6) and caused no or only transient elevations in serum transaminases. Following transient in vivo depletion of macrophages before gene transfer, significantly higher and stable FVIII expression levels were observed. Injection of only 5 × 106 HC-Ad vectors after macrophage depletion resulted in long-term therapeutic FVIII levels in the FVIIIKO and FVIIIKO-SCID mice. Intravenous injection of an HC-Ad vector into a hemophilia A dog at a dose of 4.3 × 109 IU/kg led to transient therapeutic canine FVIII levels that partially corrected whole-blood clotting time. Inhibitory antibodies to canine FVIII could not be detected, and there were no signs of hepatotoxicity or of hematologic abnormalities. These results contribute to a better understanding of the safety and efficacy of HC-Ad vectors and suggest that the therapeutic window of HC-Ad vectors could be improved by minimizing the interaction between HC-Ad vectors and the innate immune system.

scholarly journals Gene Therapy in Hemophilia: Recent Advances

2021 ◽  
Vol 22 (14) ◽  
pp. 7647
Author(s):  
E. Carlos Rodríguez-Merchán ◽  
Juan Andres De Pablo-Moreno ◽  
Antonio Liras
Keyword(s):  
Gene Therapy ◽  
Adverse Events ◽  
Factor Viii ◽  
Coagulation Factor ◽  
Cost Savings ◽  
Factor Ix ◽  
Clotting Factors ◽  
Advanced Therapies ◽  
Potential Benefits

Hemophilia is a monogenic mutational disease affecting coagulation factor VIII or factor IX genes. The palliative treatment of choice is based on the use of safe and effective recombinant clotting factors. Advanced therapies will be curative, ensuring stable and durable concentrations of the defective circulating factor. Results have so far been encouraging in terms of levels and times of expression using mainly adeno-associated vectors. However, these therapies are associated with immunogenicity and hepatotoxicity. Optimizing the vector serotypes and the transgene (variants) will boost clotting efficacy, thus increasing the viability of these protocols. It is essential that both physicians and patients be informed about the potential benefits and risks of the new therapies, and a register of gene therapy patients be kept with information of the efficacy and long-term adverse events associated with the treatments administered. In the context of hemophilia, gene therapy may result in (particularly indirect) cost savings and in a more equitable allocation of treatments. In the case of hemophilia A, further research is needed into how to effectively package the large factor VIII gene into the vector; and in the case of hemophilia B, the priority should be to optimize both the vector serotype, reducing its immunogenicity and hepatotoxicity, and the transgene, boosting its clotting efficacy so as to minimize the amount of vector administered and decrease the incidence of adverse events without compromising the efficacy of the protein expressed.

Assessing Patient Experiences with Prophylactic Treatments for Hemophilia a: Concept Elicitation for Gene Therapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-34
Author(s):  
Shawn X. Sun ◽  
Oyebimpe Olayinka-Amao ◽  
Dana DiBenedetti
Keyword(s):  
Gene Therapy ◽  
Physical Health ◽  
Pharmaceutical Company ◽  
Prophylactic Treatment ◽  
Hemophilia A ◽  
Current Therapy ◽  
Specific Patient ◽  
Hypothetical Scenario ◽  
The Impact

Background: Gene therapy for hemophilia A is designed to be a one-time infusion to deliver functional copies of the defective factor VIII (FVIII) gene, to facilitate the endogenous production of therapeutic FVIII levels. The aim is to achieve long-term protection from bleeds without the burden of regular infusions. Aims: To better understand patients' experiences of living with hemophilia A, the impact of traditional hemophilia A treatments, and patients' perceptions of the potential value of gene therapy versus traditional prophylactic treatments. Methods: Patients were identified from the database of a US rare diseases patient organization, who also recruited and screened patients for the study using materials developed by the outcomes research organization (RTI-HS) and Takeda. Adult males aged ≥18 years with a self-reported diagnosis of moderate or severe hemophilia A, who reported using factor or nonfactor prophylactic treatment and were not currently receiving treatment for inhibitors, were eligible for the study. Eligible patients provided verbal informed consent to participate in a semi-structured, 60-minute telephone interview conducted in English by 2 members of RTI-HS who did not have access to any patient-identifying information at any time during the study. Targeted questions probed perceptions of treatment burden, impact of hemophilia A on daily life, and time spent on treatment. Additionally, questions were posed to assess patients' perceptions of the impact of traditional treatments and the potential benefits they anticipate from gene therapy. Results: Nineteen patients aged 19-55 years with moderate (n = 1) or severe (n = 18) hemophilia A were interviewed. Most (16/19, 89.5%) received prophylactic FVIII therapy, (3/19, 15.8%) were receiving nonfactor prophylactic treatment, of which 1 patient also used FVIII treatment. The aspects of current or past treatments most frequently disliked by patients were lack of efficacy, frequency of infusions, intravenous administration, vein health/scar tissue, and dosing volume. Most patients expressed satisfaction with their current treatment (18/19; 94.7%), though all listed ≥1 negative treatment impact, most frequently related to difficulties with travel (13/19; 68.4%), mood/emotions (12/19; 63.2%), day-to-day activities (10/19; 52.6%), and physical health/activities (7/19; 36.8%), including having to give up or reduce particular activities because of their treatment and needing to be more cautious, especially on nontreatment days. When presented with a hypothetical scenario for gene therapy - a one-time long-acting intravenous infusion to provide a constant level of FVIII that could reduce future bleeds - all patients stated they would choose gene therapy over their current therapy, although several said they would have initial questions regarding safety, efficacy, and duration of protection. Commonly expressed reasons for preferring gene therapy (Figure 1) included fewer infusions and less worry about the need to infuse. All 19 patients said they expected to be highly satisfied with this treatment, largely because of the long-term protection from bleeds, fewer infusions, and less concern about inhibitors. The most commonly anticipated improvements were in mood/emotions (15/19; 78.9%), specifically related to reduced concern about infusions and bleed protection. Other commonly anticipated improvements included gain in time usually spent infusing (13/19; 68.4%), easier travel (12/19; 63.2%), and improved physical health and ability to perform activities (10/19; 52.6%). Conclusions: This study identified specific patient priorities, including treatment convenience, long-lasting bleed protection, frequency of intravenous infusions, and infusion volumes. The results suggest that gene therapy clinical trials should consider evaluating patient concerns in relation to the level of patient confidence in bleed protection. A study limitation is that, at the time of the survey, data on the efficacy and safety of gene therapy were limited. In the future, the study will be expanded to include a larger population of patients with hemophilia. Disclosures Sun: Takeda Pharmaceutical Company Ltd.: Current Employment. Olayinka-Amao:Takeda Pharmaceutical Company Ltd: Other: RTI-HS was contracted by Takeda Pharmaceutical Company Ltd to conduct this work; RTI Health Solutions: Current Employment. DiBenedetti:RTI Health Solutions: Current Employment; Takeda Pharmaceutical Company Ltd: Other: RTI-HS was contracted by Takeda Pharmaceutical Company Ltd to conduct this work.

scholarly journals Vedolizumab Treatment for Ulcerative Colitis in an Elderly Multimorbid Patient with Hemophilia A

2017 ◽  
Vol 11 (3) ◽  
pp. 774-779 ◽  
Author(s):  
Holger Schäffler ◽  
Astrid Huth ◽  
Georg Lamprecht ◽  
Olaf Anders
Keyword(s):  
Ulcerative Colitis ◽  
Factor Viii ◽  
Hemophilia A ◽  
Point Of View ◽  
Recurrent Bleeding ◽  
St Segment Elevation ◽  
Primary Hemostasis ◽  
Bowel Diseases ◽  
Bleeding Episodes

The treatment of inflammatory bowel diseases (IBD) can be challenging, especially in elderly multimorbid patients. Since incidence and prevalence rates of IBD are rising steadily, treatment of older patients with relevant and also rare comorbidities will be of increasing relevancy for caregivers. Here we report on a 74-year-old multimorbid patient with severe ulcerative colitis (UC) and hemophilia A. Because of the chronic active disease, therapy with a tumor necrosis factor-α inhibitor was started. He suffered from a severe infectious complication (pneumonia) under therapy with infliximab. The therapy was changed to vedolizumab, with which the patient stayed in long-term clinical and endoscopic remission. Because the patient had a non-ST-segment elevation myocardial infarction in April 2016, he received dual platelet inhibitor therapy with aspirin and clopidogrel. Because of consecutive aspirin intolerance, the therapy was changed to clopidogrel monotherapy. Although the UC was treated appropriately with vedolizumab and the patient was in endoscopic mucosal remission, recurrent bleeding episodes from multiple inflammatory pseudopolyps occurred. The bleeding episodes resolved quickly after immediate treatment with factor VIII (Kogenate®). In conclusion, we describe the first patient in the literature with UC and hemophilia A who stayed in long-term remission under therapy with vedolizumab. From our point of view, vedolizumab can be safely administered in the setting of UC and hemophilia A. Antiplatelet drugs which inhibit primary hemostasis must be used with caution in this setting. Bleeding episodes can be treated safely and effectively with factor VIII (Kogenate®).

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