Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs

Abstract

An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptide and furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy.

The Nontoxic Cell Cycle Modulator Indirubin Augments Transduction of Adeno-Associated Viral Vectors and Zinc-Finger Nuclease-Mediated Gene Targeting

“…The effect might be linked to the nuclear trafficking of the viral particles. In combination with sodium butyrate, a histone deacetylase inhibitor, ZFN-mediated gene targeting could be increased up to six-fold in a human cell line (Ellis et al, 2012). These drugs are FDAapproved, which might facilitate integration into clinical protocols for rAAV-based gene therapy; however, cytotoxic side effects on relevant primary target cells need to be investigated.…”

“…This effect has been attributed to activation of the cellular DNA damage response, which in turn is important to process the vector genome to generate a transcriptioncompetent template (Cervelli et al, 2008). The development of capsid variants optimized for cell targeting and/or trafficking (Bü ning et al, 2008;Li et al, 2008), as well as the use of compounds that do not directly damage the DNA, such as proteasome inhibitors ( Jennings et al, 2005;Monahan et al, 2010), have further improved AAV transduction efficiencies both in cultured cells (Ellis et al, 2012) and in vivo (Paulk et al, 2012). The effect might be linked to the nuclear trafficking of the viral particles.…”

Cellular Therapies: Gene Editing and Next-Gen CAR T Cells

“…Cells have been transfected by nuclease RNA, DNA or proteins through a number of methods including transfection or electroporation of nucleic acids (Urnov et al 2005 ;Porteus and Carroll 2005 ) (Lombardo et al 2007 ), adeno-associated virus (AAV) (Ellis et al 2013 ), adenovirus and lentiviral vectors (Holkers et al 2013 ). Viral methods provide effi cient delivery and nuclease expression, raising fears that over and/or prolonged expression may lead to off-target cleavage and DNA insertion.…”

Production of Transgenic Nonhuman Primates

“…Technologies such as high-throughput sequencing and gene expression microarray have accelerated the process in identifying candidate genes related to specific disease conditions. Together with novel technologies such as gene silencing by small interference RNA (siRNA) (Hitz et al, 2009;Raymond et al, 2010;Seibler et al, 2005Seibler et al, , 2007Van Pham et al, 2012;Xia et al, 2006), gene targeting by zinc finger nucleases (ZFNs) (Carbery et al, 2010;Ellis et al, 2013;Kobayashi et al, 2012;Passananti et al, 2010;Strange and Petolino, 2012), and transcription activator-like effector nucleases (TALENs) (Cermak et al, 2011;Liu et al, 2012;Mahfouz et al, 2011;Sung et al, 2012), efficient reduction of gene transcripts (knockdown) or complete knockout of the gene functions can be achieved by interfering in the transcription process by siRNA or by targeted disruption of the gene of interest using ZFN or TALEN. These novel genetic engineering tools open new opportunities in animal modeling of human genetic diseases, not only in dominant genetic disorders but also in recessive and dominant negative genetic diseases.…”