Table 1.

Limitations associated with viral vectors

Limitation	Explanation
Manufacturing	There is still lacking a robust manufacturing process to satisfy the need and produce affordable AAV-based therapeutics for patients [13]. For example, Luxturna, an AAV-based gene therapy designed to treat an uncommon type of hereditary blindness, was granted approval by the FDA in 2017. Despite being portrayed as a restorative therapy that brings back vision, it carries a high price tag of $850,000 [14]
Packaging capacity	AAV has a ~ 4.7 kb packing capacity. For SpCas9-based editing machinery, two separate vectors are needed to package SpCas9 and guide RNA, respectively, which impacts the genome editing efficiency [15–17]. Viral vectors with larger packaging capacity than AAV, such as lentivirus (~ 9.7 kb packing capacity), are more amenable to ex vivo treatments instead of in vivo ones due to biosafety concerns [18]
Genotoxicity	Retrovirus and lentivirus induce the integration of the transgene into the host genome and disrupt normal functional genes [12]. For example, in one study, 25% of patients treated with mouse Moloney retroviruses developed leukemia [17]. In addition, AAV-mediated persistent expression of CRISPR genome editors increases off-target editing effects [19]
Immunogenicity	Wild-type AAV results in priming of the immune system against the virus, with the development of both humoral and T cell immunity [20]. Pre-existing immunity to viral capsids also affects the safety and efficiency of viral vectors [21]. Besides, viral vector-induced persistent expression of CRISPR-Cas9 increases anti-Cas9 immune responses [22, 23]