Contemporary approaches for nonviral gene therapy

Abstract

Gene therapy is the manipulation of gene expression patterns in specific cells to treat genetic and pathological diseases. This manipulation is accomplished by the controlled introduction of exogenous nucleic acids into target cells. Given the size and negative charge of these biomacromolecules, the delivery process is driven by the carrier vector, of which the usage of viral vectors dominates. Taking into account the limitations of viral vectors, nonviral alternatives have gained significant attention due to their flexible design, low cytotoxicity and immunogenicity, and their gene delivery efficacy. That stated, the field of nonviral vectors has been dominated by research dedicated to overcoming barriers in gene transfer. Unfortunately, these traditional nonviral vectors have failed to completely overcome the barriers required for clinical translation and thus, have failed to match the delivery outcomes of viral vector. This has consequently encouraged the development of new, more radical approaches that have the potential for higher clinical translation. In this review, we discuss recent advances in vector technology and nucleic acid chemistry that have challenged the current understanding of nonviral systems. The diversity of these approaches highlights the numerous alternative avenues for overcoming innate and technical barriers associated with gene delivery.

Figure 1.

Generalized process for gene therapy. Nonviral vectors can be used to facilitate the delivery of DNA and RNA molecules by first mediating cellular uptake through endocytosis mechanisms. The vectors also provide protection to the genetic cargo and prompt compartmental escape as the endosome gradually acidifies. Once in the cytosol, siRNA-and miRNA-based cargo must be loaded into the RNA-induced silencing complex (RISC); whereas, mRNA must bind to cellular ribosomes to promote translation. Conversely, DNA requires translocation to the nucleus.

Figure 2.

Genome editing systems. Clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas), zinc-finger nuclease (ZFNs), and transcription activator-like effector nucleases (TALENs) are systems that can manipulate mammalian genomes with high precision and high efficiency by mediating double-strand breaks or single nicks (one strand) in a targeted sequence. The double-stand breaks are repaired by either homology-directed recombination, if a genetic donor template is available, or non-homologous end-joining.

Figure 3.

Generational changes of plasmid expression systems. Traditional plasmid DNA (pDNA) design usually contains two regions, one dedicated to plasmid propagation and the other to genetic cargo activity. However, in second generation plasmids, the vector is propagated and then processed with nucleases and ligated to remove the bacterial backbone. Alternatively, in third generation plasmids, no additional processing is required, as the plasmid propagation region is located to the intron in the transcriptional unit.

Figure 4.

CRISPR-Cas mediated plasmid self-destruct mechanism. Internal production of Cas9 and guide RNA (gRNA) target DNA in the transcriptional unit. By self-cleaving, the plasmid will moderate outcomes in the host cell.