Limits...
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

Molecular assembly of rAAV-HCR-ET3 vector particles. (a) SsDNA from rAAV-HCR-ET3 viral particles were purified, subjected to alkaline gel electrophoresis, and detected by Southern blot. (b) The molecular composition of the packaged viral genomes was determined by quantitative PCR directed against specific regions of the vector genome and normalized to the quantity of the A2 region.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4362354&req=5

fig2: Molecular assembly of rAAV-HCR-ET3 vector particles. (a) SsDNA from rAAV-HCR-ET3 viral particles were purified, subjected to alkaline gel electrophoresis, and detected by Southern blot. (b) The molecular composition of the packaged viral genomes was determined by quantitative PCR directed against specific regions of the vector genome and normalized to the quantity of the A2 region.

Mentions: To assess the effect of the oversized genome on rAAV packaging, viral ssDNA obtained from cesium chloride gradient purified rAAV-HCR-ET3 was subjected to alkaline gel electrophoresis followed by Southern blot analysis using probes directed to the A2 and C2 domain sequences of fVIII and the bovine growth hormone (BGH) polyadenylation signal sequence (Figure 2a). The rAAV-HCR-ET3 vector preparation did not contain detectable genetic material at the position expected for full-length genomes (5.9 kb). Rather, a heterogeneous smear of viral ssDNA approaching 5.0 kb was observed suggesting that the majority of viral genomic DNA was packaged as truncated fragments. It has been suggested previously that oversized transgenes, such as that encoded by rAAV-HCR-ET3, may extend beyond the capsid, exposing free 5′ ends of ssDNA on the outside of the viral particles. A comparison of Southern blot analysis of viral particles treated with DNAse prior to disruption of the viral capsid to those that were not DNAse treated failed to detect a difference in genome size, suggesting that vector genomic ssDNA does not extend beyond the viral capsid (data not shown).


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)

Molecular assembly of rAAV-HCR-ET3 vector particles. (a) SsDNA from rAAV-HCR-ET3 viral particles were purified, subjected to alkaline gel electrophoresis, and detected by Southern blot. (b) The molecular composition of the packaged viral genomes was determined by quantitative PCR directed against specific regions of the vector genome and normalized to the quantity of the A2 region.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362354&req=5

fig2: Molecular assembly of rAAV-HCR-ET3 vector particles. (a) SsDNA from rAAV-HCR-ET3 viral particles were purified, subjected to alkaline gel electrophoresis, and detected by Southern blot. (b) The molecular composition of the packaged viral genomes was determined by quantitative PCR directed against specific regions of the vector genome and normalized to the quantity of the A2 region.
Mentions: To assess the effect of the oversized genome on rAAV packaging, viral ssDNA obtained from cesium chloride gradient purified rAAV-HCR-ET3 was subjected to alkaline gel electrophoresis followed by Southern blot analysis using probes directed to the A2 and C2 domain sequences of fVIII and the bovine growth hormone (BGH) polyadenylation signal sequence (Figure 2a). The rAAV-HCR-ET3 vector preparation did not contain detectable genetic material at the position expected for full-length genomes (5.9 kb). Rather, a heterogeneous smear of viral ssDNA approaching 5.0 kb was observed suggesting that the majority of viral genomic DNA was packaged as truncated fragments. It has been suggested previously that oversized transgenes, such as that encoded by rAAV-HCR-ET3, may extend beyond the capsid, exposing free 5′ ends of ssDNA on the outside of the viral particles. A comparison of Southern blot analysis of viral particles treated with DNAse prior to disruption of the viral capsid to those that were not DNAse treated failed to detect a difference in genome size, suggesting that vector genomic ssDNA does not extend beyond the viral capsid (data not shown).

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