Advances in the Development and the Applications of Nonviral, Episomal Vectors for Gene Therapy

Abstract

Non-viral and non-integrating episomal vectors are reemerging as a valid, alternative technology to integrating viral vectors for gene therapy, due to their more favorable safety profile, significantly lower risk for insertional mutagenesis and a lesser potential for innate immune reactions, in addition to their low production cost. Over the past few years, attempts have been made to generate highly functional non-viral vectors that display long-term maintenance within cells and promote more sustained gene expression relative to conventional plasmids. Extensive research into the parameters that

A Multifaceted Approach to Optimizing AAV Delivery to the Brain for the Treatment of Neurodegenerative Diseases

“…Consideration of optimizing transgene persistence should start with identification of potential mechanisms by which transgene loss can occur. Proposed mechanisms include (1) loss of transgene expression due to immune response (Mulia et al, 2020;Konkle et al, 2021;Symington et al, 2021), (2) episomal loss due to cellular turnover without transgene replication (Lufino et al, 2008;Mulia et al, 2020), (3) epigenetic modifications resulting in gene silencing (Mulia et al, 2020). Loss of transgene expression due to the immune system has been proposed to occur either by adaptive or innate immune responses.…”

“…With AAV vector delivery, the majority of delivered transgenes will exist as an episome, rather than integrating into the host genome. This can be beneficial, as it reduces the risk of insertional mutagenesis, however, unless extra measures are taken, the transgene will not replicate with rest of the genome, overtime, transgene expression will naturally be lost (Mulia et al, 2020). Though this is a major challenge in most other organs, the highly limited neuronal replication is beneficial, in this case, and this problem may not need to be addressed when considering gene therapy for diseases predominantly involving neurons.…”

“…Epigenetic silencing poses a threat to delivered transgenes (Lufino et al, 2008;Mulia et al, 2020). There are several means to combat this.…”

“…First, it has been demonstrated that viral promoters, such as the CMV promoter, are subject to greater rates of silencing (Mulia et al, 2020) thus mammalian promoter (ideally human) should be preferentially considered. Second, specific sequences (e.g., CpG sequences) are known to be sites of gene silencing (Mulia et al, 2020;Konkle et al, 2021), minimization of these, and other sequences known to be targeted for methylation should be considered to improve transgene persistence. Finally, extrachromosomal DNA (i.e., episomes) are less subject to silencing compared integrated DNA (Lufino et al, 2008).…”

“…The major benefit of non-viral vectors is that they lack capsids, which are made up of foreign proteins and therefore are immunogenic, albeit to varying degrees (Bessis et al, 2004). Though non-viral vectors do not possess immunogenic capsids, the risk of immune response to the delivered transgene remains a major problem (Mulia et al, 2020;Ediriweera et al, 2021). Additionally, transgenes, themselves may be toxic to cells through insertion into the host's chromosomal DNA (insertional mutagenesis) (Cavazzana-Calvo and Fischer, 2007) or undesired alteration of off target protein expression (Balakrishnan et al, 2013).…”