415 Further Improvements and Applications of OPEN (Oligomerized Pool ENgineering) A Rapid, Robust, and Publicly Available Method for Engineering Customized Zinc Finger Nucleases Molecular Therapy Volu[.]
Trang 1Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
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regions from gene expression profi les in experimental arthritis and
experimentally verifi ed a panel of ten identifi ed promoters We fi rst
performed gene expression profi ling of synovial knee joint tissues
from mice with collagen-induced arthritis to identify the genes that
show at least tenfold regulation during disease progression We
then used k-means clustering to group these genes into six distinct
expression profi les Next, we introduced a fi ltering based on the
presence of a putative TATA-box between positions -32/-29 in the
upstream promoter regions of the murine genes and their human
orthologs The over-represented cis-regulatory elements in the
proximal promoters (-500/+200) that putatively govern the distinct
expression profi les were identifi ed using an algorithm that takes
advantage of spatial and phylogenetic conservation of sequences
Based on this algorithm, we constructed lentiviral luciferase reporters
containing proximal promoter regions that were predicted to be
predominantly regulated by the transcription factors NFκB (Cxcl1,
Cxcl5, Il1b), AP-1 (Mmp3, Mmp13, Timp1, Tnfaip6) or C/EBPβ
(Saa3, Chi3l1, Has1) Nine out of ten promoters were responsive to
a pro-infl ammatory stimulus in murine fi broblasts and macrophages
The promoter regions of Saa3, Cxcl1 and Mmp3 showed the strongest,
more than tenfold, increase over basal promoter activities For in vivo
validation, lentiviral reporters were injected in knee and ankle joints
of C57Bl/6 mice and the kinetics of luciferase expression during
acute infl ammation were determined by optical imaging or ex vivo
luciferase assays The promoter activities were rapidly and strongly
induced during onset of infl ammation and signifi cantly decreased
when infl ammation waned Of great value for human gene therapy,
the relative Saa3 promoter response was signifi cantly increased in
primary RA synovial fi broblasts derived from patients with a high
synovial infl ammatory gene expression profi le This study highlights
the value of a bioinformatics approach in design of transcriptionally
targeted gene therapy for disease We expect the novel vectors to be
widely applicable in both basic and translation research in human
and experimental arthritis
of a Vertebrate Chromatin Insulator
David W Emery, Karol Bomsztyk, Mari Aker
Divisions of Medical Genetics and UW Medicine Lake Union,
Department of Medicine, University of Washington, Seattle, WA.
The prototypic chromatin insulator cHS4 has proven effective
at reducing silencing chromosomal position effects in a variety of
settings Most of this barrier insulator activity has been mapped to
a 250 bp core region, as well as to several proteins that bind this
region However, recent studies from our laboratory found that an
extended 400 bp core region of the cHS4 element was necessary
to achieve full barrier insulator activity when used as a single
copy in the context of recombinant gammaretroviral and lentiviral
vectors [Aker et al., Hum Gene Ther 18:333, 2007] In more recent
studies, electrophoretic gel mobility shift assays reveled specifi c
DNA protein binding activities associated with the distal portion
of this extended core region Affi nity purifi cation and tandem mass
spectrometry studies lead to the identifi cation of one of these proteins
as poly(ADP-ribose) polymerase-1 (PARP-1), an abundant nuclear
protein that has the capacity to bind DNA through zinc fi nger motifs,
and to catalyze the addition of poly(ADP)-ribose chains to itself and
other proteins The identity of this binding activity as PARP-1 was
subsequently verifi ed by a variety of biochemical studies in vitro,
and by chromatin immunoprecipitation studies in vivo Footprinting
studies using ssDNA probes suggest that PARP-1 binding to this
extended cHS4 core region is specifi c to a unique DNA cruciform
secondary structure, rather than a primary sequence Functional
studies with gammaretroviral reporter vectors in cell lines showed
that cHS4 barrier activity was abrogated upon deletion or mutation
of the putative PARP-1 binding site, reducing the frequency of cells
expressing vector GFP 2-fold to the levels seen without the insulator Transduction studies in primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was also abrogated following treatment with a PARP inhibitor, reducing the level of vector GFP expression 2 to 7-fold to an average of 8-45 mean fl uorescent units, levels seen with the uninsulated control Finally, barrier activity
of the cHS4 element was also found to be abrogated in similar progenitor transduction studies using bone marrow from Parp1-null mice In this case, the frequency of cells expressing vector GFP from the cHS4-insulated vector decreased from an average of 51-59% in cells from normal mice to an average of 30-39% in cells from
Parp-1 null mice (compared to 23-30% for the uninsulated control) All
of these differences were statistically signifi cant PARP-1 has been shown by others to play signifi cant roles in chromatin remodeling and transcriptional control, with some evidence suggesting it may play a role in enhancer-blocking insulator activity Taken together, our studies demonstrate that binding of PARP-1 also plays a key functional role in the barrier activity of the prototypic cHS4 chromatin insulator, and helps to explain why the extended 400 bp cHS4 core exhibits more activity than smaller versions of this element
of OPEN (Oligomerized Pool ENgineering): A Rapid, Robust, and Publicly Available Method for Engineering Customized Zinc Finger Nucleases
Morgan L Maeder,1 Jing-Ruey J Yeh,2 Jonathan E Foley,1 Jizhong Zou,3 Stacey Thibodeau-Beganny,1 Linzhao Cheng,3 Randall T Peterson,2 J Keith Joung.1
1 Molecular Pathology Unit, Massachusetts General Hospital/ Harvard Medical School, Charlestown, MA; 2 Cardiovascular Research Center, Massachusetts General Hospital/Harvard Medical School, Charlestown, MA; 3 Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD.
Engineered zinc fi nger nucleases (ZFNs) can induce highly effi cient genome modifi cations in a wide variety of cell types ZFNs consist of
a customized DNA-binding zinc fi nger array fused to a non-specifi c nuclease domain ZFN-induced double-stranded DNA breaks can
be repaired by either non-homologous end-joining or homologous recombination, processes that can be used to introduce desired alterations with very high effi ciencies at or near the site of the break The development of a rapid, robust, and publicly available capability
to re-engineer the DNA-binding specifi cities of ZFNs is a critically important requirement for the practice of this technology We have recently described OPEN (Oligomerized Pool ENgineering), a user-friendly selection-based method for engineering ZFNs that accounts for the context-dependent behavior of individual zinc fi ngers (Maeder
et al., 2008) ZFNs engineered using OPEN have been used to induce highly effi cient modifi cation of numerous endogenous human, plant, and zebrafi sh genes (Maeder et al., 2008; Townsend et al., 2009; Foley
& Yeh et al., 2009) Here we describe recent alterations to the OPEN protocol which have allowed us to increase the speed of the method and to improve the scalability of selections These modifi cations, which permit the use of multi-well plates/blocks and multi-channel pipets, now enable us to perform selections for 48 target sites in less than eight weeks time We have used this higher-throughput method to engineer ZFNs for target sites in nine endogenous zebrafi sh genes To date, we have shown that many of these OPEN ZFNs can effi ciently modify their endogenous gene targets in somatic cells and that these alterations can be passed through the germline In addition, we report
on the engineering of new ZFNs targeted to the endogenous human
PIG-A gene Nonsense mutations in PIG-A are associated with
paroxysmal nocturnal hemoglobinuria (PNH) disease We have shown
that OPEN ZFNs targeted to PIG-A can stimulate gene targeting in
both human somatic and stem cells Taken together, our results enable
Trang 2Molecular Therapy Volume 17, Supplement 1, May 2009
broader and more rapid use of the OPEN ZFN selection methodology
and provide additional support for the potential use of ZFNs as an
important approach for gene therapy
Defi ciency Using a SIN Lentiviral Vector and
Human CD18 Promoter Expressing Canine CD18
Michael J Hunter,1 Laura M Tuschong,1 Cedar J Fowler,2
Everette J R Nelson,1 Tanya H Burkholder,3 Thomas R Bauer,
Jr.,1 Dennis D Hickstein.1
1 Experimental Transplantation and Immunology, National
Cancer Institute, Bethesda, MD; 2 HHMI/NIH Research Scholars
Program, Howard Hughes Medical Institute, Chevy Chase, MD;
3 Department of Veterinary Resources, National Institutes of Health,
Bethesda, MD.
Hematopoietic stem cell gene therapy would be enhanced by the
development of vectors harboring tissue-and developmental
stage-specifi c cellular promoters to express the therapeutic transgene in the
target cell population This approach would mitigate the potential
adverse effects of inappropriate tissue expression and might be
expected to reduce insertional activation of nearby oncogenes that
lead to oligoclonal hematopoiesis and leukemia To develop and test
a modifi ed promoter cassette with the features described above for
our target disease canine leukocyte adhesion defi ciency (CLAD), we
cloned portions of the human CD18 promoter (1 Kb, 776 bp, and 306
bp) into a SIN lentiviral vector upstream of the canine CD18 cDNA,
and used this vector to transduce CLAD CD34+ cells In CLAD,
defects in the leukocyte integrin CD18 result in the inability to express
CD11/CD18 heterodimers on the leukocyte surface leading to
life-threatening bacterial infections Transduction of CLAD CD34+ cells
in vitro with the SIN lentiviral vector with the 1 kb human CD18
promoter resulted in the highest percentage of CD18+ cells; nearly
15% of the CLAD CD34+ cells were CD18+ when assessed 5 days
after an overnight transduction We used the SIN lenti vector with the 1
Kb human CD18 promoter to treat two dogs with CLAD Autologous,
CLAD CD34+ cells were transduced overnight and infused following
a single, non-myeloablative dose of 200cGy total body irradiation
(TBI) The percentage of CD18+ leukocyte compartments 8 weeks
following infusion of the two dogs were comparable: dog A1, CD18+/
PMN 0.3%, CD18+/CD3+ cells 0.4%, CD18+/B-cells 0.7%, CD18+/
CD14+ cells 0.9%; dog A2, CD18+/PMN 0.5%, CD18+/CD3+
cells 0.8%, CD18+/B-cells 1.2%, CD18+/CD14+ cells 1.4% Both
treated dogs are now 4 months of age and have had correction of
the CLAD phenotype In contrast, untreated CLAD dogs succumb
to overwhelming infection within a few months of life Reversal
of the CLAD phenotype with low numbers of CD18+ neutrophils
results from selective migration of CD18+ neutrophils from the blood
into the tissues Although both CLAD dogs in this study have had a
clinical response, additional regulatory elements of the human CD18
promoter/enhancer will be required to ensure long-term reversal of
the phenotype in this and other genetic leukocyte diseases These
studies represent requisite translational studies in the development
of new vector designs for the treatment of children with the human
counterpart of CLAD, namely LAD
417 Effi cient MGMTP140K-Mediated Selection
of Long Term Repopulating Cells in a Nonhuman Primate Model
Brian C Beard,1 Grant D Trobridge,1,2 Megan L Welsh,1 Hans-Peter Kiem.1,2
1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2 Department of Medicine, University of Washington, Seattle, WA.
In vivo selection of genetically modified hematopoietic repopulating cells has many potential therapeutic applications For some applications which require relatively high levels of gene marking, such as hemoglobinopathies, in vivo selection may be required to increase initially low levels of gene-modifi ed cells Here we demonstrate effi cient post-transplantation selection of long-term hematopoietic repopulating cells using methylguanine methyltransferase (MGMTP140K) in a primate model In vivo selection was studied in macaques (M nemestrina) that received CD34-enriched cells transduced with VSVG-pseudotyped HIV-derived lentivirus vectors expressing MGMTP140K and a reporter gene green fl uorescent protein (GFP) or MGMTP140K only Two macaques were conditioned with a myeloablative dose of total body irradiation and a third macaque was conditioned with a nonmyeloablative dose of busulfan (4 mg/kg/day for 2 days) After stable engraftment monkeys were treated with O6-benzylguanine (O6BG) and BCNU Following myeloablative transplantation the monkeys transplanted with cells gene-modifi ed with a vector expressing MGMTP140K and GFP, in vivo selection was determined
by fl ow cytometry In one monkey (following 2 cycles of O6BG/ BCNU) the granulocytes rose from ∼20% to 70% and in the lymphocytes from ∼20% to 50% In the second monkey (following
a single cycle of O6BG/BCNU) the granulocytes rose from ∼25%
to 42% and in the lymphocytes from ∼12% to 22% Following nonmyeloablative transplantation and a single cycle of O6BG/BCNU the monkey transplanted with cells gene-modifi ed with the vector expressing only MGMTP140K increased overall gene marking, determined by real time (RT)-PCR, in total white blood cells rose from a provirus copy number of 0.04 (∼4% gene marking) to 0.16 (∼16% gene marking) Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed Importantly, multilineage selection of hematopoietic cells was achieved and clonality studies are underway using a combination of LAM-PCR and a modifi ed whole genome pyrosequencing approach In summary, MGMT selection is effi cient and well tolerated in macaques and these large animal studies should
be highly predictive for clinical applications and will help to further improve HSC gene therapy
Oligonucleotide Therapies
miRNAs in the CNS: Non-Allele-Specifi c Silencing
of Mutant and Wildtype Huntingtin Demonstrates Therapeutic Effi cacy in Huntington’s Disease Mice
Ryan L Boudreau,1 Jodi L McBride,1 Ines Martins,1 Shihao Shen,1
Yi Xing,1 Barrie J Carter,2 Beverly L Davidson.1
1 University of Iowa, Iowa City, IA; 2 Targeted Genetics, Seattle, WA.
RNA interference (RNAi) provides a promising therapeutic approach to treat several human diseases However, the safety of RNAi-based therapies remains a concern Previously, we compared the effi cacy and safety of short-hairpin RNA (shRNA) and artifi cial microRNA (miRNA) expression vectors in vitro and in vivo We found that shRNAs are more potent but induce toxicity in cell cultures and in mouse brain, whereas artifi cial miRNAs are expressed at lower levels and display better safety profi les We have since tested the