Gene Therapy Progress and Prospects: Development of improved lentiviral and retroviral vectors – design, biosafety, and production

Abstract

Replication defective vectors derived from simple retroviruses or the more complex genomes of lentiviruses continue to offer the advantages of long-term expression, cell and tissue specific tropism, and large packaging capacity for the delivery of therapeutic genes. The occurrence of adverse events caused by insertional mutagenesis in three patients in a gene therapy trial for X-linked SCID emphasizes the potential for problems in translating this approach to the clinic. Several genome-wide studies of retroviral integration are now providing novel insights into the integration site preferences of different vector classes. We review recent developments in vector design, integration, biosafety, and production.

Absence of ocular malignant transformation after sub-retinal delivery of rAAV2/2 or integrating lentiviral vectors in p53-deficient mice

“…46 Recent work using integrating HIV-1 vectors 14 also points to a lower risk in nondividing cells compared with dividing cells, a consequence of more favourable integration patterns associated with a substantially lower mitotic activity. Although the intraocular integration frequencies and integration patterns of rAAV2/2 vectors remain to be determined, these are also likely to be more favourable in non-dividing cells, as rAAV vectors appear to integrate preferentially at existing chromosomal breakage sites, 34 which would be expected to occur much less frequently in mitotically inactive cells.…”

Production of lentiviral vectors by large‐scale transient transfection of suspension cultures and affinity chromatography purification

“…Production of lentiviral particles can be accomplished in two different ways: by development of stable vector packaging cell lines or by transient transfection of human cells. Although the generation of several stable packaging cell lines has been achieved (Farson et al, 2001;Kafri et al, 1999;Klages et al, 2000;Kumar et al, 2003;Ni et al, 2005;Pacchia et al, 2001;Xu et al, 2001), this method is hampered by the long time required to develop a stable cell line expressing all required vector components (typically >6 months) and the lack of compatibility with cytotoxic/cytostatic transgenes or vector components that entails the need for a tight control over gene(s) expression Sinn et al, 2005). Additionally, new packaging cell lines need to be developed for each desired vector pseudotype and for each generation of improved lentiviral vectors.…”

Advances in Gene Delivery Systems

“…[10] The risk of LTR overlap-based creation of RCRs was reduced by the use of heterologous promoters and polyA signal in the packaging construct. [11] Improvement was made in the titre (number of colony-forming units per mL) by establishing stable packaging cells and by the development of transient expression systems that were capable of producing high titres. [12] The major advantage of retroviral vectors is that they integrate into the host genome, but this is a double-edged sword.…”

“…[20,21] Lentiviruses favour transgene integration near active transcription sites, while oncoretroviruses preferentially integrate at the transcription start site. [11,22] Without any accessory genes, current lentiviral vectors are considered reasonably safe, because the replication of the vectors is highly inhibited and the possibility of recombination is reduced. [22,23] Recent work showed successful gene transfer by lentiviral vectors into quiescent T and B lymphocytes for immunotherapy of several genetic dysfunctions of the haematopoietic system.…”