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1.5.1 Modifications and Implementation of Adeno-associated
Viral Vectors
AAV vectors have a long history of use in the field of gene therapy due to their
effective tropism in different cell types and lower relative cytotoxicity. With such
long history, they have also been modified in various aspects. The removal of the rep
ORF is a major modification made in recombinant AAV (rAAV) vectors. Without
this ORF, the propensity of the viral genome to integrate is decreased and the genome
is more likely to exist in the cell as an extrachromosomal episome. This decreases
the potential for insertional mutagenesis to the host genome [ 138 ]. Additionally, the
Rep protein has toxic effects on the host cell and can reduce cell viability post-AAV
infection [ 131 ]. Another modification made to some AAV vectors is the generation
of self-complementary recombinant AAV (scAAV) genomes. By decreasing the
genome size in half, the capsid can contain two complementary single stranded cop-
ies of the AAV genome. The major advantage of scAAVs is that they are much more
efficient at transduction, increasing transduction by more than 140-fold in the origi-
nal study by McCarty et al. [ 139 ]. The small size of AAVs and small packing capac-
ity is an ongoing challenge for AAV vectors used in gene therapies. One solution to
large cargoes is to split transgenes between two or more AAV vectors and co-
infection, with the transgene transcript combined after transduction into the host
cell [ 140 ]. Additionally, creating minimal versions of transgenes and regulatory ele-
ments have been proposed and attempted as a partial solution to the small DNA
capacity of AAV vectors [ 140 ]. Gene delivery using AAV vectors (and other gene
therapy vectors) falls in two broad categories: gene supplementation and gene
replacement. Gene supplementation is useful when adding additional copies of a
mutated or missing gene. Gene replacement can be used when the patient’s ineffec-
tive allele must be inactivated or replaced for normal phenotype to be restored (as is
in the case of dominant negative alleles). In the case of gene replacement, the deliv-
ery of engineerable nucleases (such as zinc-finger nuclease and CRISPR-Cas sys-
tems) to stimulate homologous recombination has been shown to be effective using
AAV vectors in animal models [ 141 – 144 ].
The host immune response to AAV vectors is a major obstacle of varying severitydepending on the method of delivery. For example, Brockstedt et al. showed that in
mice antigen-induced immune reactions in intramuscularly delivered rAAV vectors
encoding ovalbumin elicited a much reduced cytotoxic T-cell response to ovalbumin
(however the a humoral response was still present) as compared to intraperitoneally,
subcutaneously, or intravenously delivered vectors [ 145 ]. Additionally, neutralizing
antibodies have the ability to inactivate systemically delivered AAV vectors, which
can decrease transduction efficiencies in animal models and likely in human trials
as well [ 146 – 148 ]. To combat neutralizing antibody effects on transduction into
model organisms, Li et al. used in vitro directed evolution in the setting of human
serum collected from a patient to identify regions in the AAV6 capsid crucial for
evasion of neutralizing antibodies [ 149 ]. Using their results, this group generated
chimeric AAV vectors capable of improved transduction in muscle tissue [ 149 ].
J.E. DiCarlo et al.