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Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery.

Brown HC, Wright JF, Zhou S, Lytle AM, Shields JE, Spencer HT, Doering CB - Mol Ther Methods Clin Dev (2014)

Bottom Line: Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold.Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments.Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Molecular and Systems Pharmacology, Laney Graduate School, Emory University , Atlanta, Georgia, USA.

ABSTRACT
Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i) the size of the factor VIII (fVIII) transgene, (ii) humoral immune responses to fVIII, (iii) inefficient biosynthesis of human fVIII, and (iv) AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A.

No MeSH data available.


Related in: MedlinePlus

Dose finding of rAAV-HCR-ET3 in a murine model of hemophilia A. Dose finding studies were performed in male and female immune-competent hemophilia A mice. fVIII activity is presented from mice that did not form neutralizing antibodies. In male mice, (a) a long term, high dose study; (b) a medium duration, mid-dose extension study; and (c) a short term, minimum effective dose study were performed. In female mice, (d) a shorter duration dose finding study was performed. (e) A comparison between male and female mice receiving 4e12 vg/kg shows the intersex difference in fVIII levels. Baseline determination of fVIII activity in all mice tested was below the limit of detection of the assay (data not shown). N = 2–4 for all doses, error bars show one sample standard deviation. To determine the specific activity of in vivo rAAV expressed ET3, a panel of plasmas from rAAV-HCR-ET3 treated mice (open circles) were assayed for ET3 antigen level by ELISA and fVIII activity by chromogenic substrate activity assay. (f) The values obtained for each plasma were compared to a standard curve generated using highly purified recombinant ET3 diluted in hemophilia A mouse plasma (closed circles).
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fig3: Dose finding of rAAV-HCR-ET3 in a murine model of hemophilia A. Dose finding studies were performed in male and female immune-competent hemophilia A mice. fVIII activity is presented from mice that did not form neutralizing antibodies. In male mice, (a) a long term, high dose study; (b) a medium duration, mid-dose extension study; and (c) a short term, minimum effective dose study were performed. In female mice, (d) a shorter duration dose finding study was performed. (e) A comparison between male and female mice receiving 4e12 vg/kg shows the intersex difference in fVIII levels. Baseline determination of fVIII activity in all mice tested was below the limit of detection of the assay (data not shown). N = 2–4 for all doses, error bars show one sample standard deviation. To determine the specific activity of in vivo rAAV expressed ET3, a panel of plasmas from rAAV-HCR-ET3 treated mice (open circles) were assayed for ET3 antigen level by ELISA and fVIII activity by chromogenic substrate activity assay. (f) The values obtained for each plasma were compared to a standard curve generated using highly purified recombinant ET3 diluted in hemophilia A mouse plasma (closed circles).

Mentions: A dose finding study was performed to determine the ability of rAAV-HCR-ET3 to provide therapeutic levels of circulating fVIII activity in vivo. Immune-competent 8–12 week old male mice were administered a single peripheral vein injection of the vector at doses ranging from 6.2e10 to 2.0e13 vp/kg. A long term (50 weeks) study was performed on a first cohort of mice receiving high vector doses (4e12–2e13 vp/kg), while follow-up on the second cohort that received lower doses (1e12–8e12 vp/kg) was shorter (24 weeks). Finally, as the lower dose limit was not attained in the second cohort, a third cohort of mice was dosed with rAAV-HCR-ET3 at even lower levels (6.2e10–5e11 vp/kg) and was followed out to 11 weeks after AAV injection. Starting 2 weeks after vector administration, circulating plasma was assayed for fVIII activity at scheduled time points. Male mice receiving rAAV-HCR-ET3 showed dose responsive increases in circulating fVIII activity (Figure 3a–c). Supraphysiologic fVIII levels, at over >3 U/ml, were achieved at the dose of 1e13 vp/kg. For reference, the fVIII activity level observed in pooled normal human plasma is defined as 1 U/ml, while clinical hemophilia A disease classifications are severe (<1%), moderate (1–5%), and mild (5–40%) normal fVIII activity. Correction to curative levels (>0.4 U/ml or 40% normal human fVIII activity level) was achieved at doses as low as 1.0e12 vp/kg, and partial correction (>0.05 U/ml) was seen at doses as low as 5e11 vp/kg. fVIII expression was maintained throughout the duration of the experiment in all but cohorts receiving the lowest doses of vector administered and therefore defining a minimal effective dose. However, there was a trend toward activity loss over the duration of the experiment, which is consistent with gradual liver tissue turnover and loss of the episomal AAV genomes.


Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery.

Brown HC, Wright JF, Zhou S, Lytle AM, Shields JE, Spencer HT, Doering CB - Mol Ther Methods Clin Dev (2014)

Dose finding of rAAV-HCR-ET3 in a murine model of hemophilia A. Dose finding studies were performed in male and female immune-competent hemophilia A mice. fVIII activity is presented from mice that did not form neutralizing antibodies. In male mice, (a) a long term, high dose study; (b) a medium duration, mid-dose extension study; and (c) a short term, minimum effective dose study were performed. In female mice, (d) a shorter duration dose finding study was performed. (e) A comparison between male and female mice receiving 4e12 vg/kg shows the intersex difference in fVIII levels. Baseline determination of fVIII activity in all mice tested was below the limit of detection of the assay (data not shown). N = 2–4 for all doses, error bars show one sample standard deviation. To determine the specific activity of in vivo rAAV expressed ET3, a panel of plasmas from rAAV-HCR-ET3 treated mice (open circles) were assayed for ET3 antigen level by ELISA and fVIII activity by chromogenic substrate activity assay. (f) The values obtained for each plasma were compared to a standard curve generated using highly purified recombinant ET3 diluted in hemophilia A mouse plasma (closed circles).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Dose finding of rAAV-HCR-ET3 in a murine model of hemophilia A. Dose finding studies were performed in male and female immune-competent hemophilia A mice. fVIII activity is presented from mice that did not form neutralizing antibodies. In male mice, (a) a long term, high dose study; (b) a medium duration, mid-dose extension study; and (c) a short term, minimum effective dose study were performed. In female mice, (d) a shorter duration dose finding study was performed. (e) A comparison between male and female mice receiving 4e12 vg/kg shows the intersex difference in fVIII levels. Baseline determination of fVIII activity in all mice tested was below the limit of detection of the assay (data not shown). N = 2–4 for all doses, error bars show one sample standard deviation. To determine the specific activity of in vivo rAAV expressed ET3, a panel of plasmas from rAAV-HCR-ET3 treated mice (open circles) were assayed for ET3 antigen level by ELISA and fVIII activity by chromogenic substrate activity assay. (f) The values obtained for each plasma were compared to a standard curve generated using highly purified recombinant ET3 diluted in hemophilia A mouse plasma (closed circles).
Mentions: A dose finding study was performed to determine the ability of rAAV-HCR-ET3 to provide therapeutic levels of circulating fVIII activity in vivo. Immune-competent 8–12 week old male mice were administered a single peripheral vein injection of the vector at doses ranging from 6.2e10 to 2.0e13 vp/kg. A long term (50 weeks) study was performed on a first cohort of mice receiving high vector doses (4e12–2e13 vp/kg), while follow-up on the second cohort that received lower doses (1e12–8e12 vp/kg) was shorter (24 weeks). Finally, as the lower dose limit was not attained in the second cohort, a third cohort of mice was dosed with rAAV-HCR-ET3 at even lower levels (6.2e10–5e11 vp/kg) and was followed out to 11 weeks after AAV injection. Starting 2 weeks after vector administration, circulating plasma was assayed for fVIII activity at scheduled time points. Male mice receiving rAAV-HCR-ET3 showed dose responsive increases in circulating fVIII activity (Figure 3a–c). Supraphysiologic fVIII levels, at over >3 U/ml, were achieved at the dose of 1e13 vp/kg. For reference, the fVIII activity level observed in pooled normal human plasma is defined as 1 U/ml, while clinical hemophilia A disease classifications are severe (<1%), moderate (1–5%), and mild (5–40%) normal fVIII activity. Correction to curative levels (>0.4 U/ml or 40% normal human fVIII activity level) was achieved at doses as low as 1.0e12 vp/kg, and partial correction (>0.05 U/ml) was seen at doses as low as 5e11 vp/kg. fVIII expression was maintained throughout the duration of the experiment in all but cohorts receiving the lowest doses of vector administered and therefore defining a minimal effective dose. However, there was a trend toward activity loss over the duration of the experiment, which is consistent with gradual liver tissue turnover and loss of the episomal AAV genomes.

Bottom Line: Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold.Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments.Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Molecular and Systems Pharmacology, Laney Graduate School, Emory University , Atlanta, Georgia, USA.

ABSTRACT
Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i) the size of the factor VIII (fVIII) transgene, (ii) humoral immune responses to fVIII, (iii) inefficient biosynthesis of human fVIII, and (iv) AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A.

No MeSH data available.


Related in: MedlinePlus