scholarly journals 224. Helper-Dependent Adenoviral Vector Gene Therapy Mediates Long-Term Low-Level Correction of Hemophilia A in the Murine Model after Neonatal Gene Transfer

2010 ◽  
Vol 18 ◽  
pp. S86
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Murine Model ◽  
Adenoviral Vector ◽  
Hemophilia A ◽  
Low Level

Adenoviral vector–mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 100-108 ◽  
Author(s):  
Karen L. MacKenzie ◽  
Neil R. Hackett ◽  
Ronald G. Crystal ◽  
Malcolm A. S. Moore
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Adenoviral Vector ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Long Term Culture ◽  
Cd34 Cells ◽  
Infected Cells

Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level transgene expression is required. In the present investigations, we have used an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated toxicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34+ cells and primary colony forming unit–cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34+ cells. Transduction of CFU-Cs and more primitive HPCs was quantified following fluorescence activated cell sorting for green flourescence protein expression. Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more primitive cells. Although there was no apparent quantitative change in week-5 CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P < .01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU-Cs and more accurately indicated by week-5 CAFCs. These studies provide the first functional and quantitative evidence of adenoviral transduction of primitive HPCs. However, further investigations will be necessary to overcome adenoviral toxicity toward primitive HPCs before adenoviral vectors can be considered a safe option for gene therapy.

Adenoviral vector–mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 100-108 ◽  
Author(s):  
Karen L. MacKenzie ◽  
Neil R. Hackett ◽  
Ronald G. Crystal ◽  
Malcolm A. S. Moore
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Adenoviral Vector ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Long Term Culture ◽  
Cd34 Cells ◽  
Infected Cells

Abstract Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level transgene expression is required. In the present investigations, we have used an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated toxicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34+ cells and primary colony forming unit–cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34+ cells. Transduction of CFU-Cs and more primitive HPCs was quantified following fluorescence activated cell sorting for green flourescence protein expression. Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more primitive cells. Although there was no apparent quantitative change in week-5 CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P &lt; .01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU-Cs and more accurately indicated by week-5 CAFCs. These studies provide the first functional and quantitative evidence of adenoviral transduction of primitive HPCs. However, further investigations will be necessary to overcome adenoviral toxicity toward primitive HPCs before adenoviral vectors can be considered a safe option for gene therapy.

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 Sustained expression of therapeutic levels of human factor VIII in mice

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4671-4677 ◽  
Author(s):  
S Connelly ◽  
JM Gardner ◽  
RM Lyons ◽  
A McClelland ◽  
M Kaleko
Keyword(s):  
Factor Viii ◽  
Adenoviral Vector ◽  
Human Factor ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
High Dose ◽  
Specific Enzyme ◽  
Adult Mice ◽  
Human Factor Viii

Deficiency of coagulation factor VIII (FVIII) results in hemophilia A, a common hereditary bleeding disorder. Using a human FVIII-encoding adenoviral vector, Av1ALAPH81, we have demonstrated expression of therapeutic levels of human FVIII in mice sustained for more than 5 months after vector administration. Administration of a high dose (4 x 10(9) plaque-forming units [pfu]) of Av1ALAPH81 to mice resulted in a peak expression of 2,063 ng/mL of human FVIII in the mouse plasma, with levels decreasing to background by weeks 15 to 17. Normal FVIII levels in humans range from 100 to 200 ng/mL and therapeutic levels are as low as 10 ng/mL. Alternatively, administration of 8- to 80-fold lower vector doses (5 x 10(8) pfu to 5 x 10(7) pfu) to normal adult mice resulted in expression of FVIII at therapeutic levels sustained for at least 22 weeks. Detailed analysis of vector toxicity indicated that the high vector dose caused a dramatic elevation of liver-specific enzyme levels, whereas an eight-fold lower vector dose was significantly less hepatotoxic. The data presented here demonstrate that administration of lower, less toxic vector doses allow long-term persistence of FVIII expression.

scholarly journals Long-Term Transfer and Expression of the Human β-Globin Gene in a Mouse Transplant Model

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3414-3422 ◽  
Author(s):  
Harry Raftopoulos ◽  
Maureen Ward ◽  
Philippe Leboulch ◽  
Arthur Bank
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Globin Gene ◽  
High Level Expression ◽  
Hematopoietic Stem ◽  
High Level ◽  
Transplant Model

Abstract Somatic gene therapy of hemoglobinopathies depends initially on the demonstration of safe, efficient gene transfer and long-term, high-level expression of the transferred human β-globin gene in animal models. We have used a β-globin gene/β-locus control region retroviral vector containing several modifications to optimize gene transfer and expression in a mouse transplant model. In this report we show that transplantation of β-globin–transduced hematopoietic cells into lethally irradiated mice leads to the continued presence of the gene up to 8 months posttransplantation. The transferred human β-globin gene is detected in 3 of 5 mice surviving long term (>4 months) transplanted with bone marrow cells transduced with high-titer virus. Southern blotting confirms the presence of the unrearranged 5.1-kb human β-globin gene-containing provirus in 2 of these mice. In addition, long-term expression of the transferred gene is seen in 2 mice at levels of 5% and 20% that of endogenous murine β-globin at 6 and 8 months posttransplantation. We further document stem cell transduction by the successful transfer and high-level expression of the human β-globin gene from mice transduced 9 months earlier into irradiated secondary recipient mice. These results demonstrate high-level, long-term somatic human β-globin gene transfer into the hematopoietic stem cells of an animal for the first time, and suggest the potential feasibility of a retroviral gene therapy approach to sickle cell disease and the β thalassemias.

Adeno-Associated Viral Vectors for Gene Transfer and Gene Therapy

1999 ◽  
Vol 380 (6) ◽  
Author(s):  
H. Büeler
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Delivery Systems ◽  
Adeno Associated Virus ◽  
Viral Genes ◽  
Virus Recombinant

AbstractAdeno-associated virus (AAV) is a defective, non-pathogenic human parvovirus that depends for growth on coinfection with a helper adenovirus or herpes virus. Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as vectors for gene therapy. In contrast to other gene delivery systems, rAAVs lack all viral genes and show long-term gene expression

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.

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