In 2006, in an otherwise promising human clinical trial for hemophilia B, the destruction of rAAV transduced hepatocytes by cap-specific CD8+
T-cells was reported3,4
. Hauck et al., concluded that the main cause for the observed cytotoxicity was an immune response against the viral capsid proteins5
. Although potential remedies for this problem are still under debate, it is likely that the strength of a potential immune response is influenced by the amount of viral capsid proteins injected. This highlights the need for using the smallest vector dose possible and, consequently, the use of viruses with the highest achievable specific infectivity.
To date, virus preparations are evaluated by vp-to-iu ratios. Vp to iu ratios define a measure reflective of the amount of active and defective viral particles in a virus preparation and a vp-to-iu ratio of one represents a perfect virus preparation. Many parameters influence the specific infectivity of virus preparations, including the molecular mechanisms underlying replication and packaging and the purification of viral preparations. Traditionally, rAAV was purified using several rounds of cesium chloride (CsCl) gradient centrifugations. While this is a labor-intensive method that often led to varying vector quality and loss of particle infectivity, in some studies very good gcp-to-iu ratios were reported for rAAV that was purified by this method6,9
. Subsequently, faster and more reliable purification methods were developed, including the iodixanol gradient purification method7
that was used in this study. With the establishment of ELISA-based capsid quantifications12
it became possible to measure not only gcp-to-iu ratios but also vp-to-iu ratios, determining the specific infectivity of the viral preparation. Using this method, Grimm et al. reported vp-to-iu ratios for rAAV to be 1800 and above and for wtAAV to be 42 and above12
in crude, non-purified virus preparations. These findings implied that viral preparations were contaminated with empty or otherwise defective particles. Therefore, to determine accurately the specific infectivity of a given vector preparation, the physical viral particle, the genome-containing particle and the infectious particle titer of a given vector preparation need to be known.
In this report, we determined these three parameters using well-established AAV titration methods14
. The resulting vp-to-iu ratio of several wtAAV2 and rAAV2 virus preparations produced and purified with identical, standard methods could be determined. We found that preparations of wtAAV2 can approach not only vp-to-gcp ratios of one but, more importantly, can also approach vp-to-iu ratios of one ( and ). These results suggest that the “ceiling” for the specific infectivity of AAV-based vectors is close to a vp-to-iu ratio of one, i.e. the ratio of a perfect virus. To our knowledge, to date, no virus has been discovered that has a vp-to-iu ratio approaching the perfect ratio of one. Employing the same methods, we then compared the wtAAV2 preparations to rAAV2 preparations containing either the GFP or the mCherry transgene. In contrast to wtAAV2, rAAV2 preparations showed a vp-to-iu ratio of 53 to 124 ( and ). The difference between wild-type and recombinant viruses raises the question as to what the underlying mechanisms are, that result in suboptimal rAAV2 preparations. For example, it would be possible that 123 out of 124 viral particles do not contain a genome. However, consistent with earlier observations10,11
, our electron microscopy results (), as well as the Q-PCR and amino acid analysis titers (), show that approximately half of the viral particles contain a genome.
In order to reduce the proportion of empty or otherwise defective particles and, hence to improve the infectivity of rAAV preparations, two questions need to be answered: First, what causes the difference in packaging efficiency and specific infectivity of wild-type versus recombinant viruses and second, what can be changed in order to minimize the amount of empty or otherwise defective viral particles in rAAV preparations? The removal of empty capsids from genome-containing vectors has recently been addressed. Qu et al.11
published an improved and scalable method for the purification of rAAV vectors that can remove empty particles from the vector preparation yielding a vp-to-gcp ratio of 1.2 for rAAV. Unfortunately, it was not reported how many of these full particles were infectious.
A potentially even more promising approach than more advanced purification methods to minimize the fraction of empty or defective particles is to improve the production methods in order to prevent the formation of these particles in the first place. To do this, it is important to understand the reason(s) for the differences in packaging/replication efficiency between wtAAV2 and rAAV2. There are at least four hypotheses: (i) a cis
-acting DNA element within the wtAAV2 genome can act as an as yet unidentified replication/packaging signal. Several groups presented evidence for the existence of such an element. In 2000, Tullis and Shenk identified a 1688 base pair cis
-acting element that spans a large part of the rep
gene and they demonstrated the importance of this element for virus replication. Once this region was deleted from the wtAAV2 genome, its replication efficiency was similar to rAAV2 vectors21
. Ward et al., confirmed this observation by deleting various parts of the left portion of the wtAAV genome, which lead to a reduction in virus production efficiency22
. The Salvetti group identified a different cis
-acting replication element (CARE) at nucleotide 250-304 in wtAAV2 that is required for rep
-dependent replication in the absence of the ITRs23,24
. However, in the context of rAAV with ITRs, no effect on replication and encapsidation was reported when this element was deleted22
. Taken together, a cis
-acting replication/packaging element that can improve the overall quality of rAAV preparations—if existent—remains to be identified conclusively. The system provided in this report, i.e. using wtAAV as a benchmark for virus infectivity, could be used to prove the existence of such an element. (ii) rather than a cis
-acting element, the complete wtAAV genome might be required to ensure the necessary DNA-capsid interactions. (iii) Expression of the rep
gene in trans
(rAAV)—as opposed to in cis
(wtAAV)—might result in a decrease of replication and/or encapsidation. Ward et al., found a significant reduction in virus production when parts of the cap
gene were deleted or a stop mutation was introduced in the cap
. It was concluded that, as suggested previously25,26
, the provision of cap
does not sufficiently support the DNA replication-encapsidation link. (iv) The size of the genome to be packaged might have an important impact on the quality of the virus. It was previously shown that genomes smaller than 3500 base pairs are defective in accumulation of single stranded DNA21
. The genomes of our rAAV2-GFP and rAAV2-mCherry are 4329 and 4323 bases long, respectively. Because these genomes are similar in length to wt-AAV2 (4679 bases), they should allow the synthesis of similar amounts of single stranded DNA when compared to wtAAV.
In conclusion, our data suggest a functional difference between wild-type and recombinant AAV particles. We provide a system, namely the comparison of the specific infectivity of a given rAAV preparation to that of wtAAV2 produced and purified in an identical manner, that in the future could be employed to dissect the molecular differences that underlie the formation of different wtAAV2 and rAAV2 particles. While we propose the use of wtAAV as a benchmark to compare the quality of rAAV vectors, we do not mean to imply that the perfect quality determined in vitro will translate into perfect transduction in vivo. Similarly, comparison of rAAV vectors of alternative serotypes or other AAV isolates as well as vectors purified by different methods will have to be standardized to their respective wtAAV counterpart independently. However, insights gained by such comparison studies could result in improved vector design and lead to the requirement for significantly lower viral vector doses than are currently used in clinical studies thereby likely not only improving the efficiency of gene transfer but also reducing the risk of a deleterious immune response.