Molecular analysis of vector genome structures after liver transduction by conventional and self-complementary adeno-associated viral serotype vectors in murine and nonhuman primate models

Abstract

Vectors based on several new adeno-associated viral (AAV) serotypes demonstrated strong hepatocyte tropism and transduction efficiency in both small- and large-animal models for liver-directed gene transfer. Efficiency of liver transduction by AAV vectors can be further improved in both murine and nonhuman primate (NHP) animals when the vector genomes are packaged in a self-complementary (sc) format. In an attempt to understand potential molecular mechanism(s) responsible for enhanced transduction efficiency of the sc vector in liver, we performed extensive molecular studies of genome structures of conventional single-stranded (ss) and sc AAV vectors from liver after AAV gene transfer in both mice and NHPs. These included treatment with exonucleases with specific substrate preferences, single-cutter restriction enzyme digestion and polarity-specific hybridization-based vector genome mapping, and bacteriophage phi29 DNA polymerase-mediated and double-stranded circular template-specific rescue of persisted circular genomes. In mouse liver, vector genomes of both genome formats seemed to persist primarily as episomal circular forms, but sc vectors converted into circular forms more rapidly and efficiently. However, the overall differences in vector genome abundance and structure in the liver between ss and sc vectors could not account for the remarkable differences in transduction. Molecular structures of persistent genomes of both ss and sc vectors were significantly more heterogeneous in macaque liver, with noticeable structural rearrangements that warrant further characterizations.

FIG. 1.

Comparison of improvement in liver transduction by AAV serotype vectors with a self-complementary (sc) genome over a conventional single-stranded (ss) genome; abundance of vector genomes detected in mouse liver and biological activities of the vectors on a per-genome basis at various stages of gene transfer. (A) Enhancement of liver transduction by AAV vectors with an sc genome over an ss genome in mouse liver. Four- to 6-week-old male C57BL/6 mice were treated via portal vein injection with AAV serotype 2, 7, and 8 vectors (10¹⁰ or 10¹¹ genome copies [GC]/animal) carrying ss and sc genomes and expressing choriogonadotrophic hormone (rhCG) under the control of a CMV-enhanced chicken β-actin promoter. Sera were collected from the animals on days 3 and 90 after vector administration for an ELISA-based rhCG assay. Transgene expression data are presented as the fold increase in sc over ss genomes. (B) Comparison of biological activities of vectors on a per-genome basis between different AAV serotypes and genotypes at different stages of gene transfer. Only data from the animals that received high-dose vectors (10¹¹ GC/mouse) were analyzed. (C) Comparison of genome abundance in mouse livers that had received sc and ss vectors; comparison done on days 3 and 90 after vector delivery. Total cellular DNAs were extracted from mouse livers and subjected to either real-time PCR quantification or DNA hybridization analysis after BamHI and HindIII digestion to drop an internal 787-bp fragment. An 800-bp rhCG cDNA fragment was used as a probe for hybridization.

FIG. 2.

Characterization of circular molecular forms of AAV vector genomes in mouse liver. (A) Mouse liver DNAs from the animals received AAVCBrhCG vectors (10 μg each) were either not treated (U) or treated with EcoRV, a restriction enzyme that does not cut within the vector genomes (N), or with Plasmid-Safe exonuclease (P), at 37°C overnight. M, molecular weight marker. The samples were then subjected to gel electrophoresis on a 1% agarose gel followed by DNA hybridization analysis with an rhCG cDNA probe. (B) The PS-treated samples were also subjected to rolling circular linear amplification (RCLA). One-twentieth of each RCLA product was cut with NotI, a single-cutter in the vector genome, and analyzed on ethidium bromide (EtBr)-stained 1% agarose gels. PSR, Plasmid-Safe resistant; PSS, Plasmid-Safe sensitive; dsc, double-stranded circle; sdsmc, supercoiled double-stranded monomeric circle; ssg, single-stranded genome; lc, linearized circle; dsl, double-stranded linear genome.

FIG. 3.

DNA hybridization analysis of molecular forms of vector genomes in mouse liver DNA after digestion with a single-cutter restriction enzyme. Liver DNAs were extracted on days 3 and 90 from animals receiving various AAV serotype vectors with various genotypes and digested with either MluI (ss) or HpaI (sc). (A) DNA hybridization analysis with a 5′ probe (H probe). (B) DNA hybridization analysis with a 3′ probe (T probe). ldsc, linearized double-stranded circle; dsl, H: double-stranded linear genome, head; dsl, T: double-stranded linear genome, tail; ssg, single-stranded genome.

FIG. 4.

DNA hybridization analysis of molecular forms of vector genomes in the liver of macaques infused with ss and sc AAV vectors. Ten micrograms each of cellular DNAs from macaque livers harvested at the end of study were subjected to various exonuclease and restriction endonuclease digestions. (A) Detection of PS-resistant vector genomes by DNA hybridization analysis. Macaque liver DNAs from the animals that received AAVCBrhCG vectors (10 μg each) were either not treated (U) or treated with EcoRV, a restriction enzyme that does not cut within the vector genomes (N), or with Plasmid-Safe exonuclease (P) at 37°C overnight. M, molecular weight marker. (B and C) Diagnosis of molecular forms of vector genomes in macaque liver by various single- and double-cutter restriction enzyme digestions and DNA hybridization analysis. Liver DNAs from sc recombinant AAV (scrAAV)-treated animals (scrAAV7, AJ7R and AJ28; scrAAV8, AT3E and AT3F) were digested with two different single cutters individually, either XhoI or SacI [(B) and (C), respectively]. H–H, head-to-head; H–T, head-to-tail. (D) Liver DNAs from sc vector-treated animals were digested with two sets of single cutters together (XhoI + SacI). Those single cutters cut at the ends of vector genomes. The blots were hybridized with 5′ and 3′ probes separately.

FIG. 4.

DNA hybridization analysis of molecular forms of vector genomes in the liver of macaques infused with ss and sc AAV vectors. Ten micrograms each of cellular DNAs from macaque livers harvested at the end of study were subjected to various exonuclease and restriction endonuclease digestions. (A) Detection of PS-resistant vector genomes by DNA hybridization analysis. Macaque liver DNAs from the animals that received AAVCBrhCG vectors (10 μg each) were either not treated (U) or treated with EcoRV, a restriction enzyme that does not cut within the vector genomes (N), or with Plasmid-Safe exonuclease (P) at 37°C overnight. M, molecular weight marker. (B and C) Diagnosis of molecular forms of vector genomes in macaque liver by various single- and double-cutter restriction enzyme digestions and DNA hybridization analysis. Liver DNAs from sc recombinant AAV (scrAAV)-treated animals (scrAAV7, AJ7R and AJ28; scrAAV8, AT3E and AT3F) were digested with two different single cutters individually, either XhoI or SacI [(B) and (C), respectively]. H–H, head-to-head; H–T, head-to-tail. (D) Liver DNAs from sc vector-treated animals were digested with two sets of single cutters together (XhoI + SacI). Those single cutters cut at the ends of vector genomes. The blots were hybridized with 5′ and 3′ probes separately.

FIG. 5.

Summary of molecular fate of AAV vector genomes in mouse (A) and macaque (B) liver, based on the results of single-cutter digestion and DNA hybridization study. The dotted circles mark the regions in the persistent vector genomes that might have been deleted or rearranged.