Improving Viral Vectors for Gene Targeting in Gene Therapy

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Title: Improving Viral Vectors for Gene Targeting in Gene Therapy
Author: Ellis, Brian Lee
Abstract: Over 10 ,000 monogenic diseases in the world affect one out of every hundred live births (WHO ) . Gene targeting is a term that is used describe the manipulation of genetic material , either by adding a gene in a specific locus , creating a mutation at a specific locus , or correcting a gene at a specific locus . Here , unless otherwise noted , we will use the term to describe the correction of a gene with a homologous piece of donor genetic material whereby a mutant gene that causes monogenic disease is essentially replaced by a wild type copy through homologous recombination . Thus , gene targeting is inherently safer than classic gene therapy , where a gene is randomly introduced into the genome and can cause insertional mutagenesis . Although the rates of homologous recombination are low when simply delivering a donor substrate (1 in a million ) , creating a deoxyribonucleic acid (DNA ) double -stranded break in or around the gene of interest using a nuclease , increases the rate of gene targeting 30 ,000 -50 ,000 fold . The delivery of the nuclease and donor substrate to these cells is one of the major hurdles in achieving this type of therapy . However , for classic gene therapy there have already been many clinical trials using viral vehicles for gene delivery . One problem with using a virus for gene therapy is the low titer associated with some types of virus , in particular , lentivirus . In the first part of this dissertation , this problem is addressed by showing that the addition of caffeine during viral production can increase titer up to 8 -fold . Besides lentivirus , other viruses , like Adeno -associated virus (AAV ) have been used in clinical trials . There are nine AAV serotypes , but the most -well characterized is AAV2 . Because there are situations where AAV is to be used in cells that cannot be transduced with AAV2 , it is essential to know which serotype best infects the desired cell type . The second part of the dissertation describes a comprehensive survey of the ability of AAV1 -9 and one engineered serotype to transduce primary and immortalized cells from human , mouse , hamster , and monkey origin . Overall , the results show that AAV1 and AAV6 transduce the most cell types at the highest efficiencies . Though gene targeting has been achieved using the homing endonuclease I -Sce in AAV2 , targeting has never been achieved using two zinc -finger nucleases (ZFNs ) in any AAV serotype . This is significant because the recognition site for I -Sce is not found in the human genome , while ZFNs are designed to specifically bind in or around a gene of interest . Based on the results from the AAV survey and the advantage of ZFNs , we created an AAV6 virus that carried the genetic information for both ZFNs and donor substrate for gene targeting in cells containing a GFP gene targeting system . We also created an AAV6 virus that carried the donor substrate alone . The third part of this dissertation reveals that dual infection at the optimal multiplicities of infection for both AAV viruses can achieve targeting efficiencies of ~3 % , which is ~3 -fold higher than by lipofection . Furthermore , we show that the addition of the proteasome inhibitor , MG132 , increases the gene targeting level an additional 2 -fold . This data suggests that AAV is a great choice for gene therapy by gene targeting . Chapters 3 -5 within this body of work make significant contributions to the gene therapy field . The work and the contributions will be described in each section respectively as well as summarized in the last chapter .
URI: http : / /hdl .handle .net /2152 .5 /837
Date: 2011-02-01

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Improving Viral Vectors for Gene Targeting in Gene Therapy. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /837 .

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