Supplementary AAV Notes

AAV Intracellular Trafficking

The events and processes that regulate trafficking of AAV particles into the nucleus are still not fully understood. It has been speculated that the viruses enter cells through receptor-mediated endocytosis, and internalised virions escape from endosomal degradation by a low pH-dependent process. Additionally, given its small particle size, it has been suggested that AAV particles can access the nucleus, through the nuclear pore complex (NPC) dependent and independent pathways.

Biological function of AAV Capsid Protein

Although it is relatively easy to see AAV capsid protein involvement in target cell contact, when discussing this topic, it should be noted that capsid proteins are also thought to be critical in intracellular trafficking of AAV, such as endosomal escape, uncoating and nuclear entry. All these steps eventually determine the AAV tropism.

Other AAV Production Methods

The development of scalable transfection-independent methods for rAAV production has been pursued lately, due to the requirement for large amounts of highly purified vector particles to conduct large animal studies and human clinical trials. One proposed method involves the generation of packaging cell lines with stably integrated AAV rep and cap genes. The establishment of effective, high-titer producer cell lines has proven difficult, due to the inhibitory effects of Rep proteins on cell growth. Nonetheless, improvements in the control of rep expression through the development of stringent inducible gene expression systems may overcome these effects.

rAAV Purification Method Developments

In parallel to new rAAV production platforms, insights into AAV biology could lead to significant improvements in the purity of vectors based on AAV2 along with other serotypes. Specifically, knowledge on AAV receptor usage could potentially lead to the implementation of scalable affinity column chromatography purification schemes. A more broadly applicable column chromatography procedure, based on the ion-exchange principle, has recently been developed for the purification of rAAV2, rAAV4 and rAAV5 particles. However, compared to the traditional CsCl ultra-centrifugation method (the gold standard of virus preparation) the quantity, as well as quality consistency prepared through these procedures remain to be thoroughly tested, before applying it broadly.

AAV Vector DNA Persistence

The expression of genes delivered by rAAV can persist for quite a long time in organs/tissues without notable toxicity accumulation. However, it should be noted that this stability is not caused by the transgene DNA insertion into the AAVSI locus. Due to the absence of rep gene products, the rAAV DNA is not targeted to the host genome. Moreover, because rAAV vectors lack those viral genes altogether, the molecular fate of the DNA once in the nucleus is dependent on host cell activities, though a role for the virion capsomers (the only viral proteins that come with the rAAV) cannot be ruled out. For the transgene expression to occur, the single-stranded rAAV genomes need to be converted into a transcriptionally functional, double-stranded template. A recent study indicates that free (i.e., unpackaged) single-stranded rAAV genomes have a very transient presence in the target cell, because the majority is recognized by host enzymes as damaged DNA and degraded. The big question is: what mechanism(s) exist through which (1) the single-stranded genome DNA are converted to double-stranded DNA, and (2) the long-term expression of rAAV-delivered transgene is achieved?

Regarding the single- to double-stranded rAAV conversion, several hypotheses have been proposed. One of them is dubbed plus/minus annealing pathway. It should be noted that, the single-stranded AAV genomes with sense (plus) and anti-sense (minus) orientations are packaged equally well. Therefore, it is possible that, inside targeted cells, the formation of double-stranded AAV genome DNA involves the annealing of 2 single-stranded genome molecules with opposing polarities. However, the conversion of single-stranded DNA into double-stranded DNA form contribute to, but couldn’t completely explain the persistent expression of AAV-delivered gene, since the linear, double-stranded DNA could be easily degraded by cellular mechanism(s). Now the question is: how the rAAV DNA persistence is maintained?

It was recently proposed that, after the single- to double-stranded DNA conversion, the duplex rAAV genomes, through either intra- or inter-molecular recombination at the ITRs, lead to circular forms or linear concatemers. In addition, the circular episomes can also evolve into high-molecular- weight concatamers. This hypothesis is consistent with observation that circular episomal forms of recombinant AAV vectors that have been isolated form in vivo transduced tissues. The balance between linear versus circular forms is, at least in part, regulated by a complex cellular machinery, including DNA-dependent protein kinase (DNA-PK). It has been postulated that free double-stranded rAAV DNA ends are substrates for the cellular double-stranded break repair machinery responsible for free-ended DNA removal. It is the circular forms, or its concatamers, are thought to be responsible for the stable maintenance of transgene expression. Though intriguing, this as well as other proposed hypotheses, need to be substantiated with more studies.

In short, though rAAV could maintain relatively long-term expression of transgene, the exactly molecular mechanism(s) through which it is achieved is far from clear. However, it is not through the integration of the recombinant rAAV DNA into the AAVSI site, as previously assumed. Better understanding of this phenomenon could lead to the development of new versions of rAAV vectors.

Recent Progress in Vector Technologies

(1) The small packaging capacity of AAV particles (about 4.7 kb) is considered one of the main limitations of rAAV vectors. As discussed above, incoming linear rAAV genomes can form concatamers in target cells through intermolecular recombination at their free ends. This phenomenon has been successfully exploited to assemble in target cells large genetic messages through the joining of two independently transduced rAAV genomes, each of which encompasses a portion of a large transcriptional unit. mRNA molecules encoding a functional protein are generated from the rAAV DNA head-to-tail heterodimers by splicing out the AAV ITR sequences from the primary transcripts. Although this split gene strategy allows expression of almost double-sized transgenes after rAAV-mediated gene delivery, its efficiency is significantly lower than that observed with a single control vector encoding the full-length transgene. Both vectors have to transduce the same cell and only heteroconcatamers with a head-to-tail organization will give rise to a functional full-length gene product.

(2) Another development in rAAV design is the so-called “self-complementary AAV vectors” (scAAV). The scAAV approach builds on the ability of AAV to package replicons with half the size of wild-type DNA in the form of single-stranded dimeric genomes with an inverted repeat configuration. In the target cell, these self-complementary molecules can readily fold back into double-stranded forms without the need for de novo DNA synthesis or for the annealing of sense and antisense strands. Ultimately, regardless of the mechanism(s) at play, scAAV lead to enhanced formation of transcription competent double-stranded genomes, thus improving the expression kinetics and yields of vector-encoded products. The main disadvantage of this approach is the need to limit the size of the transgenes that can be delivered to approximately half the length of the already small AAV genome. For instance, if a transgene is under the control of a CMV-driven expression cassette, the maximal gene of interest allowed is about 1.0-1.2Kb, significantly lower than that of the ssAAV – about 3.5Kb.

(3) Another recent development is in the mutation of AAV capsid proteins.  Lately, it was shown that, by mutating some tyrosine residues on the surface of AAV2 Cap protein, the infection efficiency of rAAV2 is significantly increased. This is probably due to the inhibition of ubiquitination of AAV capsids followed by proteasome-mediated degradation. It will be interesting to see whether the increased infectivity observed in some cell types could be repeated in other cell types in vitro, as well as in various tissue/organs in vivo.

In Conclusion

Insights from AAV biology have been instrumental in this process and are expected to continue to be the main catalyst behind further development and efficacious deployment of rAAV. Given the reported problem with gene therapy studies using other viral vectors, such as adenovirus and lentivirus, rAAV has been at the forefront of all vector systems that aim at safe and sustained transgene expression in vivo. Because of the enormous effort and resources being put into this area of research, it is conceivable that we should see exciting advances for using rAAV or its derivative(s) as a gene delivery tool, both for basic studies and clinical development.

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