Search

Recombinant adeno-associated virus serotype 9 (rAAV9) vectors show robust in vivo transduction by a systemic approach. It has been proposed that rAAV9 has enhanced ability to cross the vascular endothelial barriers. However, the scientific basis of systemic administration of rAAV9 and its transduction mechanisms have not been fully established. Here, we show indirect evidence suggesting that capillary walls still remain as a significant barrier to rAAV9 in cardiac transduction but not so in hepatic transduction in mice, and the distinctively delayed blood clearance of rAAV9 plays an important role in overcoming this barrier, contributing to robust cardiac transduction. We find that transvascular transport of rAAV9 in the heart is a capacity-limited slow process and occurs in the absence of caveolin-1, the major component of caveolae that mediate endothelial transcytosis. In addition, a reverse genetic study identifies the outer region of the icosahedral threefold capsid protrusions as a potential culprit for rAAV9's delayed blood clearance. These results support a model in which the delayed blood clearance of rAAV9 sustains the capacity-limited slow transvascular vector transport and plays a role in mediating robust cardiac transduction, and provide important implications in AAV capsid engineering to create new rAAV variants with more desirable properties.

It has been recently shown that recombinant adeno-associated virus serotype 8 (rAAV8) is a robust alternative serotype vector that overcomes many of the limitations of rAAV2 and transduces various tissues efficiently and globally through systemic vector administration. AAV9 is a serotype newly isolated from human tissues, but our knowledge of the biology of rAAV9 in vivo is currently limited. Here, we demonstrate by a series of comprehensive side-by-side experiments with rAAV8 and 9 vectors delivered via different routes or at various doses in mice that rAAV9 vectors share the robustness of rAAV8, i.e., (1) very high liver transduction efficiency irrespective of whether vectors are administered intravascularly or extravascularly and (2) substantial transduction in the heart, skeletal muscle, and pancreas by peripheral vein injection. Importantly, rAAV9 transduced myocardium 5- to 10-fold higher than rAAV8, resulting in over 80% cardiomyocyte transduction following tail vein injection of as low as 1.0 x 10(11) particles per mouse. Thus rAAV9, as well as rAAV8, is a robust vector for gene therapy applications and rAAV9 is superior to rAAV8 specifically for cardiac gene delivery by systemic vector administration.

Mammalian cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) sequence informations. These fragments were subsequently used to screen a rat liver cDNA library, yielding a 1573-bp clone. Expression of DNA fragment containing either of two possible open reading frames (nucleotide numbers 97-1431 and 148-1431) of the isolated clone in yeast led to the accumulation of gamma-linolenic acid in the presence of exogenous linoleic acid. In this system, the addition of alpha-linolenic acid also resulted in the accumulation of its Delta-6 desaturated product whereas dihomo-gamma-linolenic acid failed to be a substrate. These results indicate that the protein encoded by the rat cDNA is Delta-6 fatty acid desaturase, and the first 17 amino acids corresponding to the coding region 97-147 of the clone are not required to function in yeast.

rAAV8 vectors transduce various tissues with extremely high efficiency in neonates and infants. Many applications of rAAV8 vector injection into prenatal or neonatal mice have been proposed to treat various disorders. Intraperitoneal or intravascular injection of rAAV8 vector into neonatal mice results in widespread efficient transduction in heart and skeletal muscles, which can stably persist even at adult ages. In contrast, the liver is transduced efficiently with rAAV8 only in the neonatal period, and liver transduction substantially declines thereafter. Despite the limited efficacy in neonatal liver transduction, it is important to understand the biology of rAAV8 vectors in the liver following neonatal administration because 1) the neonatal liver is an ideal target organ for gene transfer in many instances; and 2) substantial vector genome dissemination to the liver is likely to occur in non-hepatic gene transfer. In order to substantially understand the biology of rAAV8 injected into neonates, we non-injected or injected intraperitoneally C57BL/6 and 44Bri mice with AAV8-EF1|[alpha]|-nlslacZ vector at birth, and followed up total 69 animals. 44Bri is an HBV transgenic strain that develops chronic liver injury at |[thksim]|3 months of age, causing continuous liver regeneration. At the age of 110|[ndash]|179 days, the mice received AAV9-LSP (liver-specific promoter)-hFIX vector via the tail vein. 17 days post rAAV9 injection, half of the mice underwent 2/3 partial hepatectomy (PHx), and all the mice were sacrificed 6 weeks after PHx. The rAAV9 vector served as an internal control for PHx in rAAV8-injected animals. Xgal staining of the liver sections revealed sparsely scattered single blue hepatocytes and occasional clusters comprising 2|[ndash]|20 blue hepatocytes. Non-parenchymal blue cell foci were also found in many liver sections analyzed. Several relatively large blue hepatocyte clusters comprising of supposedly 10e2|[sim]|10e4 hepatocytes were found in one wild type and two 44Bri mice after PHx, suggesting that rAAV8-transduced cells in these foci might have had growth advantage over surrounding non-transduced hepatocytes. hFIX expression from the rAAV9 and rAAV9 genome copy numbers in the liver substantially dropped (over 90%) by PHx, while rAAV8 vector copy number drops were significantly smaller than those of rAAV9 vector (27-92% vector copy number drops for wild type and 73% drop-869% increase for 44Bri mice). This indicates that, although a significant portion of rAAV8 vector genomes were extrachromosomal, vector genomes were apparently integrated into chromosomes, with frequency of at most 2.7 vector genome copy/cell in our study. Thus, rAAV8 injected at birth integrates into chromosomes in the liver, stably transducing both parenchymal and non-parenchymal hepatic cells sparsely. In parallel to this experiment, we are following up 69 non-injected and 117 rAAV8-injected (at birth) wild type and 44Bri male mice over 400 days to investigate procarcinogenic potential of rAAV8 in mice. A preliminary result in this study will be also presented.

Top of pageAbstract DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role in rAAV vector transduction. Recently, it has been reported that rAAV vector genome integration into chromosomes is substantially increased in scid mouse muscles and livers (PNAS 98: 4084, 2001; PNAS 101:2112, 2004). In scid livers, they observed that >40% of transgene expression and >50% of vector genomes remained after partial hepatectomy (PHx). However, we have not been able to confirm their observations. Rather we have found that liver regeneration is impaired and slowed in scid mice, particularly in older animals, resulting in insufficient dilution of episomal genomes after PHx. In order to revisit this subject, we have established a method that can directly quantify integrated vector genomes by Southern blot, and performed an experiment with a careful monitoring of liver regeneration after PHx. Since we found that animal age is a critical factor for liver regeneration in scid mice, we prepared relatively young age-matched 8-week-old C57BL/6 and C57BL/6 scid male mice. We injected these mice with AAV2 or 8-ISceI.AO3 vector as indicated in Table 1 (n=5|[ndash]|6 per group). The AAV-ISceI.AO3 vectors had an ISceI site in their vector genomes. With these vectors and ISceI digestion, we could clearly separate episomal and integrated vector genomes in transduced tissues, therefore quantify the amount of integrated vector genomes by Southern blot. 7 weeks after injection, 3 mice from each group underwent PHx, and all the mice were sacrificed 8 weeks after PHx. Careful liver weight measurement revealed that liver regeneration in scid mice was impaired by a factor of 0.88. Vector genome copy numbers at and 8 weeks after PHx are summarized in Table 1. The results demonstrate that vector genome copy numbers substantially dropped by 71|[ndash]|90% in scid mouse livers, and there was no significant difference in the reduction of vector genome copy numbers between B6 and scid mice. This sharply contrasts with the previous observations by Song et al. In addition, Southern blot analysis that can quantify integrated vector genomes revealed no detectable integrated vector genomes in any of the liver, skeletal muscle and kidney. Thus we conclude that scid phenotype does not substantially increase rAAV vector genome integration frequency at a detectable level in vivo.

Top of pageAbstract We reported last year that double-stranded (ds) linear rAAV vector genomes with a |[ldquo]|dog bone|[rdquo]| structure (i.e., a dumbbell-shaped ds rAAV genome with both terminal hairpin loops closed) emerged in mouse hepatocytes in the absence of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs, i.e., scid phenotype) but not in the presence of DNA-PKcs (i.e., wild type) when a rAAV8 vector was injected via the tail vein at a very high dose, 7.2 |[times]| 10e12 vector genome (vg) per mouse. We and others have shown that such an altered fate of rAAV vector genomes in murine liver was not observed when rAAV2 vectors were injected at a regular dose. Based on this observation, we have hypothesized that 1) AAV-ITRs are recognized by cells as |[ldquo]|closed|[rdquo]| hairpins whether or not the ITRs have a 5' or 3' DNA end (i.e., |[ldquo]|open|[rdquo]| ITRs as opposed to the |[ldquo]|closed|[rdquo]|), and 2) hairpin opening of |[ldquo]|closed|[rdquo]| ITRs by a specific cellular endonuclease(s) is the very first step that triggers all vector genome recombinations toward the formation of ds circular monomer genomes and concatemers, and vector genome integration into chromosomal DNA in vivo. If AAV-ITR hairpins are not resolved, 5' and 3' DNA ends in vector genomes are ligated with each other intramolecularly by a cellular ligase activity, forming the |[ldquo]|dog bone|[rdquo]| structure, and further intermolecular recombinations through the ITRs would not occur. In the present study, we further investigated the |[ldquo]|dog bone|[rdquo]| genomes as a tool to understand the mechanisms of AAV-ITR hairpin opening and rAAV vector genome recombinations in mice. Consequently, we have elucidated the following: (1) |[ldquo]|dog bone|[rdquo]| genomes emerged in scid liver only when rAAV8 vectors were injected at doses of 1.8 |[times]| 10e12 or higher, at which vector copy numbers in the livers were > 800 ds-vg/dge (ds vector genomes per diploid genomic equivalent); (2) rAAV vectors delivered to scid liver by rAAV2 vectors never resulted in accumulation of |[ldquo]|dog bone|[rdquo]| genomes no matter how much vectors were injected; (3) in non-hepatic scid tissues such as heart, muscle and kidney, |[ldquo]|dog bone|[rdquo]| genomes accumulated at a much lower vector genome load (|[sim]|2 ds-vg/dge); (4) hepatocyte cell cycling recruited a supplementary AAV- ITR hairpin opening activity that did not exist in a quiescent state, facilitating vector genome recombinations through the AAV-ITRs. These observations demonstrate that AAV-ITR hairpin opening in non-hepatic tissues largely depends on the DNA-PKcs-associated cellular endonuclease activity, while in the liver, the pathways of AAV-ITR hairpin opening are redundant and cell cycle dependent. Based on the recent discovery of DNA-PKcs/Artemis pathway for DNA double-strand break repair together with our observations, we propose that a cellular endonuclease, presumably Artemis, plays a crucial role in AAV-ITR hairpin opening and subsequent vector genome recombinations in concert with DNA-PKcs. We are currently investigating the role of Artemis in this process using Artemis deficient cells.

Recombinant AAV(rAAV) vectors are promising vehicles for achieving stable transduction in vivo. We have demonstrated that, in contrast to rAAV2 vectors, which have limited transduction efficiency in many tissues, rAAV8 vectors can transduce all the hepatocytes, cardiomyocytes and skeletal myofibers throughout the body, and a majority of pancreatic acinar cells by peripheral vein injection at a high dose, in mice. AAV serotype 9 has been recently isolated from non-human primate tissues and recombinant vectors based on this serotype have been shown to transduce various tissues with high efficiency. In order to characterize the behavior of rAAV9 vectors in mice, we injected mice with various rAAV9 vectors via several different routes and analyzed transgene expression and tissue distribution. First, we produced a liver specific promoter-driven human factor IX (hFIX)-expressing rAAV9 vector, AAV9-hFIX16, and injected adult C57BL/6 male and female mice via 4 different routes; i.e., portal vein (PV), tail vein (TV), intraperitoneal (IP) and subcutaneous (SC) injections at a dose of 8.0|[times]|10e10 vg/mouse. For comparison, we also injected mice with AAV8-hFIX16 at the same dose. Liver transduction efficiency was monitored by measuring hFIX levels in the plasma. Two weeks post-injection, PV injection with the rAAV9 vector (9PV), 9TV and 9IP achieved comparable hFIX levels of 350-400 |[mu]|g/ml, which was slightly higher than the mice injected with the rAAV8 vector via PV (8PV). SC injection achieved approximately half of the levels with both AAV9 and AAV8 vectors, but still expressed over 100 |[mu]|g/ml of hFIX in plasma at this dose. Liver transduction in females was lower than males by |[sim]|30% with both serotypes as seen with rAAV2 vectors in this mouse strain. Interestingly, TV and IP injections of the rAAV9 vector showed slower kinetics of hFIX expression compared to the PV injection, even though any of these routes achieved a comparable level of expression 2 weeks-post injection. Such distinct expression kinetics depending on the route of vector administration was not observed with rAAV8 vector. In the second experiment, we injected C57BL/6 mice with rAAV9 vectors expressing a lacZ marker gene; i.e., AAV9-EF1|[alpha]|-nlslacZ or AAV9-CMV-lacZ via TV at two different doses, 3.0|[times]|10e11 and 1.8|[times]|10e12 vg/mouse (n=2 per group, total 8 mice for 4 groups). Development of anti-|[acirc]| galactosidase antibody was monitored by ELISA. The antibody was not detected in any of the 8 mice 7 days post-injection, but detected in 3 of 4 mice injected with AAV9-CMV-lacZ 10 days post-injection, when all the 8 mice were sacrificed for tissue distribution analysis. Liver transduction efficiency with the CMV vector was 3% at 3.0|[times]|10e11 and reached 90% at 1.8|[times]|10e12 vg/mouse. The tissue distribution pattern with rAAV9 vectors was similar to that with rAAV8 vectors, in that, in addition to the liver, they transduced heart, skeletal muscle and pancreatic acinar cells with high efficiency. Of note is that rAAV9 had greater transduction efficiency in the heart than rAAV8, and 3.0|[times]|10e11 vg/mouse was enough to transduce all the cardiomyocytes via TV injection of rAAV9. Thus, rAAV9, as well as rAAV8, is a robust vector for gene therapy applications.

rAAV vectors integrate into host chromosomal DNA infrequently. However, we still may need to consider the possibility of adverse effects caused by rAAV vector integration because 1) rAAV2 vector integrations in animals accompany host chromosomal deletions or translocations, and occur preferentially in or near gene regulatory sequences; and 2) 3 to 4 % integrations occur in or near cancer-related genes. In order to further address this issue and understand the biology of rAAV vectors in vivo, we investigated rAAV8 vector integration in normal and DNA-PKcs-deficient scid mice. Scid mice were chosen because DNA-PKcs has been shown to be involved in rAAV vector genome processing. Here, we developed a novel strategy for isolation of a large number of rAAV proviruses directly from vector-transduced animal tissues with high efficiency and reliability, and without any selective pressure. The salient features of the strategy are: 1) the use of a shuttle rAAV8 vector carrying an ISceI site and AmpR/Ori (AO) cassette, 2) physical separation of integrated and extrachromosomal vector genomes following ISceI digestion of tissue DNA; 3) removal of unwanted genomic DNA by digestion with a DNA conformation-dependent nuclease; 4) mixing the tissue DNA with yeast genomic DNA at 1:1 ratio at the beginning of the procedure, with yeast DNA serving as a tag sequence to monitor for undesired intermolecular recombination that may arise during the procedure. We injected normal and scid mice with 7.2|[times]|10e12 vg of this rAAV8 shuttle vector via the tail vein, and made rAAV8 integration provirus libraries from transduced mouse liver DNA. Transformation of bacteria with |[sim]|0.3 |[mu]|g liver DNA generated over a thousand colonies in each library. Initial sequencing results of 121 and 69 clones from normal and scid mouse libraries demonstrated that 23 (19%) and 52 (75%) of the sequenced clones carried rAAV8 vector-cellular DNA junctions, respectively. Importantly, we have not seen any intermolecular recombination between rAAV8 vector and yeast genomes, establishing the high reliability of this strategy. A preliminary analysis suggested that integration sites in scid mice are more dispersed than in normal mice. In addition, we found that a majority of rAAV8 genomes in scid mouse liver form extrachromosomal double-stranded |[ldquo]|dog bone|[rdquo]| structures, i.e., with both terminal hairpin loops closed. Such dog bone forms are present at undetectable levels in rAAV2-injected mouse livers, but may represent intermediates toward various vector forms. Thus the isolation of a large number of rAAV proviral genomes from quiescent somatic cells in animals is feasible by our novel strategy, and high-throughput rAAV integration site analyses in normal and scid mouse tissues and detailed characterization of the dog bone forms will provide new insights into the mechanisms of rAAV vector transduction and integration in animal tissues.