K137R Mutation on Adeno-Associated Viral Capsids Had Minimal Effect on Enhancing Gene DeliveryIn Vivo

Abstract

The adeno-associated viral (AAV) vector has emerged as an attractive vector for gene therapy applications. Development of AAV vectors with enhanced gene transduction efficiency is important to ease the burden of AAV production and minimize potential immune responses. Rational mutations on AAV capsids have gained attention as a simple method of enhancing AAV transduction efficiency. A single-amino acid mutation, K137R, on AAV1 and AAV8 was recently reported to increase liver transgene expression by 5-10-fold. To determine whether the same mutation on other AAV serotypes would result in similar gene enhancement effects, K137R mutants were generated on AAV7, AAV8, and AAV9, and their effects were evaluated in vivo. Two reporter genes were utilized: the nuclear LacZ gene driven by the cytomegalovirus promoter and the luciferase gene driven by the CB promoter. Surprisingly, we found no difference in luciferase gene expression in the liver or other tissues using either the wild-type AAV8 capsid or AAV8-K137R. LacZ gene expression in the liver by AAV8-K137R was about onefold higher than that of wild-type AAV8. However, no difference was found in other tissues, such as skeletal muscle and cardiac muscle. In addition, no difference was found in transgene expression with either AAV7-K137R or AAV9-K137R mutants. Our results indicated that the K137R mutation on AAV7, AAV8, and AAV9 had minimal to no effect on transduction efficiency in vivo.

Site-Directed Mutagenesis of Surface-Exposed Lysine Residues Leads to Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes In Vivo

“…We also concur with Xiao and colleagues 33 and their conclusion that additional variables, such as the sensitivity of the reporter genes, the vector titers, and the vector potency, could also be major factors. These results, nonetheless, underscore the need to exercise caution in relying on, and interpreting data from, experimental rodent models, as well as the need for pursuing additional studies using non-human primate models to delineate the role of each of the critical Y, S, T, and K residues to get at the underlying molecular mechanism of the observed differences in the transduction efficiency of these mutant AAV8 vectors in various tissues in general and hepatocytes in particular.…”

“…32,33 Whereas Jayandharan and colleagues 32 reported that a single lysine-mutant (K137) of scAAV8-EGFP vector increased the transduction efficiency by 11-fold in hepatocytes in C57BL/6J mice, Xiao and colleagues, 33 using the same K137R-AAV8 mutant vectors expressing either the LacZ or the …”

“…Subsequently, the same group also reported that a single lysine-mutant (K137R) of scAAV8-EGFP vector increased the transduction efficiency by 11-fold, 32 a claim that was not substantiated by a report by Xiao and colleagues. 33 In view of these discrepancies, we …”

Single point mutation in adeno-associated viral vectors -DJ capsid leads to improvement for gene delivery in vivo

“…Our study on Y to F mutants of both AAV8 and AAV-DJ also displayed minimal enhancement of gene expression in vivo (to be published). Furthermore, Qiao et al also obtained negative results of K137R on AAV8 [ 34 ], which were reported to enhance gene expression by 5 to 10 fold in liver [ 15 ]. These inconsistent results may be caused by using different reporters contributing to various measurements of transduction efficiency, incorrect vector titers, dilutions, and/or inconsistent vector potency from batch to batch.…”

Adeno-associated virus-mediated gene therapy in cardiovascular disease

“…From a clinical point of view, this issue is very significant because the presence of neutralizing antibodies against the AAV serotype limits the potential clinical candidates for the gene therapy [15▪]. Over the past years, significant effort has been put into the development of AAV variants that circumvent the effect of neutralizing antibodies [14,16]. Recently, a chimeric AAV vector developed by capsid reengineering was shown to effectively transduce the heart for the first time in a clinically relevant large animal model [17▪].…”