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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: Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications.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.

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

Viral vector design and in vitro expression. The 5.86 kb rAAV-HCR-ET3 genome encodes the high expression bioengineered fVIII molecule ET3, which consists of porcine fVIII sequences in the A1 and ap-A3 domains and human sequence in the A2, C1, and C2 domains. The ET3 transgene is under the control of a liver-specific hAAT promoter/ApoEHCR enhancer sequence, and termination is governed by a bovine growth hormone poly adenylation signal (bGHPA). (a) The genome is flanked by AAV2 ITRs on both the 5′ and 3′ ends. fVIII activity was measured in conditioned medium of HepG2 cells that were transiently transfected with rAAV-HCR viral expression vectors encoding ET3 or the non-bioengineered BDD hfVIII construct HSQ. fVIII mRNA levels were measured by quantitative reverse transcription PCR. (b) For comparison of biosynthetic efficiency, the ratio of fVIII activity production to fVIII mRNA transcripts per cell is displayed. (c) Plasma fVIII activity was measured in hemophilia A mice that were hydrodynamically injected with rAAV-HCR viral expression plasmid encoding ET3 or HSQ. Baseline fVIII levels were determined to be below the limit of detection for all mice (data not shown). All error bars show one sample standard deviation, N = 3 for in vitro studies and 3–4 for in vivo studies.
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fig1: Viral vector design and in vitro expression. The 5.86 kb rAAV-HCR-ET3 genome encodes the high expression bioengineered fVIII molecule ET3, which consists of porcine fVIII sequences in the A1 and ap-A3 domains and human sequence in the A2, C1, and C2 domains. The ET3 transgene is under the control of a liver-specific hAAT promoter/ApoEHCR enhancer sequence, and termination is governed by a bovine growth hormone poly adenylation signal (bGHPA). (a) The genome is flanked by AAV2 ITRs on both the 5′ and 3′ ends. fVIII activity was measured in conditioned medium of HepG2 cells that were transiently transfected with rAAV-HCR viral expression vectors encoding ET3 or the non-bioengineered BDD hfVIII construct HSQ. fVIII mRNA levels were measured by quantitative reverse transcription PCR. (b) For comparison of biosynthetic efficiency, the ratio of fVIII activity production to fVIII mRNA transcripts per cell is displayed. (c) Plasma fVIII activity was measured in hemophilia A mice that were hydrodynamically injected with rAAV-HCR viral expression plasmid encoding ET3 or HSQ. Baseline fVIII levels were determined to be below the limit of detection for all mice (data not shown). All error bars show one sample standard deviation, N = 3 for in vitro studies and 3–4 for in vivo studies.

Mentions: The rAAV vector design was based on constructs previously used to express the human coagulation factor IX transgene from liver tissue.15 The ET3 transgene, which consists of human fVIII sequences in the A2, C1, and C2 domains and porcine fVIII sequences in the A1 and ap-A3 domains, or alternatively, a BDD hfVIII transgene with a 14 amino acid linker (SQ), designated HSQ, was cloned into an AAV expression cassette controlled by a liver-specific ApoEhepatic control region (HCR)/human alpha-1 antitrypsin (hAAT) enhancer/promoter and flanked by AAV2 inverted terminal repeats (ITRs) (Figure 1a). Complete amino acid alignment of HSQ and ET3 amino acid sequences reveals 91 percent identity (Supplementary Figure S2). We previously have shown that the increased expression of ET3 is conferred through enhanced posttranslational processing of the nascent fVIII peptide.13 To determine if improved biosynthetic efficiency was retained by the AAV-HCR-ET3 expression cassette, an in vitro transfection experiment utilizing the human hepatocellular carcinoma HepG2 cell line was performed. AAV-HCR-ET3 and AAV-HCR-HSQ expression plasmids were transiently transfected into HepG2 cells for assessment of fVIII transcript levels and secreted fVIII activity. Although cells transfected with AAV-HCR-ET3 plasmid contained greater numbers of fVIII mRNA transcripts per cell than those transfected with AAV-HCR-HSQ (850 ± 39 versus 284 ± 69), this 3-fold differential in mRNA level could not account for the >20-fold differential in fVIII activity observed in the conditioned medium (0.70 ± 0.24 units (U)/ml for ET3, and 0.034 ± 0.01 U/ml for HSQ). Thus, AAV-HCR-ET3 transfected HepG2 cells demonstrated sevenfold higher levels of fVIII production per mRNA transcript than the AAV-HCR-HSQ transfected cells suggesting that post mRNA biosynthetic efficiency of ET3 expression, presumably endoplasmic reticulum to golgi transit, is the primary determinant of high level expression in the context of AAV based liver-directed expression (Figure 1b). However, we cannot rule out that increased transcriptional efficiency or mRNA stability may further contribute to the enhanced expression of ET3 compared to HSQ. To further examine the finding of enhanced expression of ET3, an in vivo comparison of the two vector-transgene designs by hydrodynamic injection of the expression plasmids was performed. In this experimental system, again the AAV-HCR-ET3 expression plasmid conferred 20-fold higher plasma levels of fVIII activity than AAV-HCR-HSQ expression plasmid further supporting the claim of enhanced production of ET3 compared to HSQ (Figure 1c, Supplementary Table S3).


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)

Viral vector design and in vitro expression. The 5.86 kb rAAV-HCR-ET3 genome encodes the high expression bioengineered fVIII molecule ET3, which consists of porcine fVIII sequences in the A1 and ap-A3 domains and human sequence in the A2, C1, and C2 domains. The ET3 transgene is under the control of a liver-specific hAAT promoter/ApoEHCR enhancer sequence, and termination is governed by a bovine growth hormone poly adenylation signal (bGHPA). (a) The genome is flanked by AAV2 ITRs on both the 5′ and 3′ ends. fVIII activity was measured in conditioned medium of HepG2 cells that were transiently transfected with rAAV-HCR viral expression vectors encoding ET3 or the non-bioengineered BDD hfVIII construct HSQ. fVIII mRNA levels were measured by quantitative reverse transcription PCR. (b) For comparison of biosynthetic efficiency, the ratio of fVIII activity production to fVIII mRNA transcripts per cell is displayed. (c) Plasma fVIII activity was measured in hemophilia A mice that were hydrodynamically injected with rAAV-HCR viral expression plasmid encoding ET3 or HSQ. Baseline fVIII levels were determined to be below the limit of detection for all mice (data not shown). All error bars show one sample standard deviation, N = 3 for in vitro studies and 3–4 for in vivo studies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Viral vector design and in vitro expression. The 5.86 kb rAAV-HCR-ET3 genome encodes the high expression bioengineered fVIII molecule ET3, which consists of porcine fVIII sequences in the A1 and ap-A3 domains and human sequence in the A2, C1, and C2 domains. The ET3 transgene is under the control of a liver-specific hAAT promoter/ApoEHCR enhancer sequence, and termination is governed by a bovine growth hormone poly adenylation signal (bGHPA). (a) The genome is flanked by AAV2 ITRs on both the 5′ and 3′ ends. fVIII activity was measured in conditioned medium of HepG2 cells that were transiently transfected with rAAV-HCR viral expression vectors encoding ET3 or the non-bioengineered BDD hfVIII construct HSQ. fVIII mRNA levels were measured by quantitative reverse transcription PCR. (b) For comparison of biosynthetic efficiency, the ratio of fVIII activity production to fVIII mRNA transcripts per cell is displayed. (c) Plasma fVIII activity was measured in hemophilia A mice that were hydrodynamically injected with rAAV-HCR viral expression plasmid encoding ET3 or HSQ. Baseline fVIII levels were determined to be below the limit of detection for all mice (data not shown). All error bars show one sample standard deviation, N = 3 for in vitro studies and 3–4 for in vivo studies.
Mentions: The rAAV vector design was based on constructs previously used to express the human coagulation factor IX transgene from liver tissue.15 The ET3 transgene, which consists of human fVIII sequences in the A2, C1, and C2 domains and porcine fVIII sequences in the A1 and ap-A3 domains, or alternatively, a BDD hfVIII transgene with a 14 amino acid linker (SQ), designated HSQ, was cloned into an AAV expression cassette controlled by a liver-specific ApoEhepatic control region (HCR)/human alpha-1 antitrypsin (hAAT) enhancer/promoter and flanked by AAV2 inverted terminal repeats (ITRs) (Figure 1a). Complete amino acid alignment of HSQ and ET3 amino acid sequences reveals 91 percent identity (Supplementary Figure S2). We previously have shown that the increased expression of ET3 is conferred through enhanced posttranslational processing of the nascent fVIII peptide.13 To determine if improved biosynthetic efficiency was retained by the AAV-HCR-ET3 expression cassette, an in vitro transfection experiment utilizing the human hepatocellular carcinoma HepG2 cell line was performed. AAV-HCR-ET3 and AAV-HCR-HSQ expression plasmids were transiently transfected into HepG2 cells for assessment of fVIII transcript levels and secreted fVIII activity. Although cells transfected with AAV-HCR-ET3 plasmid contained greater numbers of fVIII mRNA transcripts per cell than those transfected with AAV-HCR-HSQ (850 ± 39 versus 284 ± 69), this 3-fold differential in mRNA level could not account for the >20-fold differential in fVIII activity observed in the conditioned medium (0.70 ± 0.24 units (U)/ml for ET3, and 0.034 ± 0.01 U/ml for HSQ). Thus, AAV-HCR-ET3 transfected HepG2 cells demonstrated sevenfold higher levels of fVIII production per mRNA transcript than the AAV-HCR-HSQ transfected cells suggesting that post mRNA biosynthetic efficiency of ET3 expression, presumably endoplasmic reticulum to golgi transit, is the primary determinant of high level expression in the context of AAV based liver-directed expression (Figure 1b). However, we cannot rule out that increased transcriptional efficiency or mRNA stability may further contribute to the enhanced expression of ET3 compared to HSQ. To further examine the finding of enhanced expression of ET3, an in vivo comparison of the two vector-transgene designs by hydrodynamic injection of the expression plasmids was performed. In this experimental system, again the AAV-HCR-ET3 expression plasmid conferred 20-fold higher plasma levels of fVIII activity than AAV-HCR-HSQ expression plasmid further supporting the claim of enhanced production of ET3 compared to HSQ (Figure 1c, Supplementary Table S3).

Bottom Line: Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications.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.

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