Targeted Integration of Foreign DNA Into a Defined Locus on Chromosome 19 in K562 Cells Using AAV-Derived Components

Abstract

Targeted integration of foreign DNA is ideal for gene therapy, particularly when target cells such as hematopoietic cells actively divide and proliferate. Adeno-associated virus (AAV) has been shown to integrate its genome into a defined locus, AAVS1 (19q13.3-qter). The inverted terminal repeat (ITR) and Rep proteins are responsible for this site-specific integration, and a system has been developed that delivers a gene preferentially into AAVS1 by using these components of AAV. We examined whether this system could be applied to gene transfer into K562 cells. Two rep expression plasmids were tested, 1 driven by the cytomegalovirus (CMV) promoter (pCMVR78) and the other under the translational control of an internal ribosome entry site (pMGiR78) with mouse mammary tumor virus promoter. K562 cells were cotransfected with a rep plasmid and a plasmid containing a neo gene flanked by the ITRs. G418-resistant clones were isolated and analyzed by Southern blot analysis and fluorescence in situ hybridization (FISH). Southern blot analysis suggested AAVS1-specific integration of the neo gene in 6 (35%) of 17 clones when K562 cells were transfected with pMGiR78 by lipofection. FISH located the neo gene on chromosome 19 in 5 of these 6 clones (29%). Eight (32%) of 25 clones obtained by electroporation with pCMVR78 had the neo gene at AAVS1, according to Southern blot analysis, and 4 of these 8 clones (16%) were positive according to FISH analysis. These results suggest that site-specific integration of foreign DNA can be achieved at a significantly high rate in human hematopoietic cells using the AAV components.

Second Generation Adeno-Associated Virus Type 2-based Gene Therapy Systems with the Potential for Preferential Integration into AAVS1

“…Similar results were seen using cationic liposome-mediated transfection [Kogure et al, 2001;Lamartina et al, 1998;Satoh et al, 2000;Tsunoda et al, 2000;Young, Xiao, and Samulski, 2000] or electroporation [Kogure et al, 2001].…”

“…Preferential integration, using plasmid-based systems has been demonstrated in cell lines derived from human embryonic kidney cells, human erythroid leukemia cells, a human cervical carcinoma and a human hepatocarcinoma [Balagué, Kalla, and Zhang, 1997;Kogure et al, 2001;Lamartina et al, 1998;Pieroni et al, 1998;Rinaudo et al, 2000;Satoh et al, 2000;Surosky et al, 1997;Young, Xiao, and Samulski, 2000]. The major problem with transfection is that it tends to be much less efficient than virus particle-mediated gene transfer, especially for delivery to intact organs.…”

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies

“…To this end, AAV-based site-directed integration systems may be useful, because Rep proteins from AAV facilitate the integration of the viral genome into a specific locus (termed AAVS1) in chromosome 19. 26 This locus is one of the safe locations in the human genome for efficient and safe transgene expression. 27 Ammar et al 28 demonstrated that fusion constructs consisting of Rep and Tol2 transposase integrated marker genes from Tol2 transposon donor plasmids near the AAVS1 locus, which may offer a safer transposon-based gene delivery system for human gene and cell therapy.…”

Gene- and cell-based therapeutics for type I diabetes mellitus

“…The list indirectly demonstrates that no 'ideal' vector yet exists. New technology, including small interference RNA (siRNA), 31 adeno-associated virus inverted terminal repeat (AAV ITR)-based plasmids, 32,33 novel classes of lentivirus (equine infectious anemia virus-EIAV; feline immunodeficiency virus-FIV), [34][35][36][37][38][39][40] lentivirus-herpesvirus hybrids and other viral vectors is in development, but their efficiency has yet to be reported in the context of Figure 1 Multistep process of insulitis. During ontogeny, a population of thymocytes whose TCR recognize b-cell-specific antigens are either not deleted in the thymus, or fail to be tolerized subsequently, in the periphery.…”