Scholarly article on topic '1156. Methods for Assembling Complex Vectors without Sub-Cloning'

1156. Methods for Assembling Complex Vectors without Sub-Cloning Academic research paper on "Biological sciences"

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Academic research paper on topic "1156. Methods for Assembling Complex Vectors without Sub-Cloning"

1154. Targeting In Vivo Gene Correction Using Designed DNA-Binding Proteins

Fyodor D. Urnov,1 Jeffrey C. Miller,1 Carl C. Pabo.1 'Sangamo BioSciences, Richmond, CA.

Progress in understanding the molecular etiology of human monogenic disorders has not been followed by an increase in efficiency of therapeutic intervention strategies. However robust our ability to genotype individuals with cystic fibrosis, sickle cell anemia, lysosomal storage disorders, or deficiencies in genes for blood clotting factors, therapeutic options are limited to enzyme replacement therapy, or non-curative management of symptoms. We describe our recent studies toward a novel and efficient in vivo gene correction approach to this problem that brings together three technical advances: (i) massive potentiation of homologous recombination (HR) and gene correction/conversion in mammalian cells via the introduction of a double-stranded break (DSB) at the mutant locus (M. Jasin, Trends Genet. 12: 224; 1996); (ii) the ability of cleavage domains from certain type IIS restriction endonucleases, such as Fokl, to create a DSB at a given locus when fused to a heterologous DNA-binding domain (Kim and Chandrasegaran, PNAS 91: 883; 1994); (iii) the ability to design high affinity, single locus-specific DNA-binding domains against any sequence of choice using engineered zinc finger proteins (Rebar et al., Nature Medicine 8:1427; 2002). Most human genetic disorders are caused by hypomorphic or null mutations in genes, and restoration of even a single wild-type allele to the cell can be curative. In practice, however, the human genome is resistant to gene correction or replacement by HR. As a consequence, therapeutic transgenes carrying wild-type sequences integrate at nonnative loci, and this misintegration results in aberrant genome behavior (Hacein-Bey-Abina, NEJM 348: 255; 2003), or in the failure of the transgene to express properly (Ellis, Clin. Genet. 59: 17; 2001). Introduction of a DSB at the target locus activates a potent repair pathway, during which genome integrity is restored by replacing the DNA sequence at the DSB with genetic information from a homologous DNA molecule (Johnson and Jasin, Biochem. Soc. Trans. 29: 196; 2001). With this approach, genetic information from an engineered episome efficiently and accurately replaces the homologous chromosomal sequence. In model systems, mutated reporter genes have been engineered to carry recognition sites for endonucleases, whose action was then shown to potentiate HR and gene conversion from a wild-type donor sequence by > 3 orders of magnitude. Reporter genes have been constructed with recognition sites for designed ZFP-based DNA-binding domains, and chimeras of such ZFPs with the Fokl cleavage domain have been shown to massively potentiate HR in model systems (Bibikova et al., MCB 21: 289). Recent technological advances have implemented an idea proposed in 1991 (Pavletich and Pabo, Science 252: 809; 1991) — to use the C2H2 ZFP motif as a scaffold to design novel-specificity DNA binding domains. Extensive published data demonstrate the versatitility, potency, and specificity of designed ZFPs as regulators of mammalian genomes in vivo (Urnov et al., EMBO Rep. 3: 610; 2002). Use of such designed ZFPs as fusions with endonuclease domains to target DSBs to any position of choice in the human genome offers an unprecedented opportunity to enable locus-specific, high-efficiency gene correction therapeutic strategies for human monogenic disorders.

Full-time employee of Sangamo BioSciences, Inc.

1155. External Regulation of Lentiviral Vectors by Targeting Transcriptional Activation Loop

Geetanjali Sachdeva,1 Jean E. Cho,1 Suresh K. Arya.1,2 'Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, United States; 2Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Bethesda, MD, United States.

With the knowledge of human genome in hand and accelerating pace of annotation, the need for efficient gene delivery for experimental and therapeutic purposes will become acute. With parallel progress in the ability to manipulate stem cells and because of the uncommon ability of lentiviral vectors to transduce non dividing cells, these vectors will come to occupy a prominent place in gene transfer. The translational potential of lentiviral mediated gene transfer will be enhanced by acquiring the ability to regulate them in vivo by signal supplied from outside. We propose to search for 'drug-like' molecules that will achieve vector regulation from outside. The availability of complex chemical libraries and high throughput screening technology makes this exercise possible. The library search is based on the premise that molecules exist, or can be made to exist, that modulate all known biological functions. As a proof of principal that regulatory pathway can be targeted, we have chosen to target the Tat-mediated transciptional activation loop of lentiviruses. We aim to obtain drug-like molecules that will be substitutes for Tat, that is, achieve activation of LTR in the absence of Tat. Note that intent here is to upregulate HIV expression and not down regulate it as is the case for the research for antiviral drugs. To discriminate between global effect and specific modulation, studies with hTERT promoter will be carried in parallel. Thus, the search aims both at a viral target and a cellular target. A small molecule acting on hTERT promoter will be valuable for cancer gene therapy. We have created lentiviral transfer vectors with the GFP indicator gene where its expression is under the control of the viral LTR. They in addition contain IRES-linked puromycin resistance gene in a bicistronic configuration for cell selection. Similar clones have been created for the hTERT promoter. For creating stably-transfected cell lines for cell-based screening assays, a number of cell lines have been tested to meet the following criteria: no basal viral LTR activity, responsive to added Tat - genetically or extracellularly introduced, responsive to known hTERT modulators, deficient in MDR function, and stable maintenance of the introduced gene cassette. Protocol optimization for screening with the 'training set' and 'diversity set' are in progress, which will be followed by mounting a 'screening campaign'.

1156. Methods for Assembling Complex Vectors without Sub-Cloning

Lance Johnson,1 James Williams,1 Clague Hodgson.1 'Genetics, Nature Technology Corporation, Lincoln, NE, United States.

Increasingly complex vectors needed for gene therapy often require multiple sub-cloning steps. In order to facilitate the ordered, directional assembly of genes and vectors, we devised new vectors for a modular approach, in which each DNA fragment is assigned two unique, non-palindromic address tags. This method relies upon the ability of class IIS restriction enzymes to digest at a distance from the enzyme site. Blunt DNA fragments (synthetic dsDNA; PCR amplicons; or blunt restriction fragments) are cloned into a novel vector, PWiz-Bang2.0™, and the class IIS enzyme AarI (a seven base cutter), or an AarI isoschizomer, is used to cleave the DNA, leaving a four base, 5'-overhang. The fragments are recovered and are ligated in a single reaction, producing the desired construct. The 4-base pendant termini ligated with high fidelity. Three isolates each of two different plasmids containing six ligated fragments, all yielded the desired constructs. The ability to sequence input

Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy

fragments before gene construction provides assurance that the final constructs will not contain mutations caused by faulty oligonucleotide primers (common), or from PCR enzymes (rare). In theory, the use of unique, non-palindromic 4-base termini permits up to 121 fragments to be joined unambiguously together. The method is useful for combinatorial chemistry (see abstract #451508), because it permits pools of fragments to be joined together with single-nucleotide precision. The process requires oligonucleotide primers of high integrity, together with a high-fidelity polymerase enzyme (such as Pfu DNA polymerase) that does not add extra bases (if gene amplification is used). Primary uses of the technology include the construction of synthetic genes from dsDNA oligonucleotides, as well as complex viral and non-viral vectors needed for gene therapy.

All authors have an equity interest in Nature Technology Corporation

Advances in Clinical Gene Therapy

1157. INGN 241 (Ad-mda7) Induces Widespread Apoptosis and Activates the Immune System in Patients with Advanced Cancer

Sunil Chada,1 Casey Cunningham,2 Yuan Zhang,3 Dan Su,3 Abner Mhashilkar,1 Suhendan Ekmekcioglu,4 Elizabeth Grimm,4 Deborah Wilson,1 James Merritt,1 Keith Coffee,1 John Nemunaitis,2 Alex W. Tong.2,3

'Introgen Therapeutics, Inc, Houston, TX; 2US Oncology, Dallas, TX; 3Cancer Immunology Research Laboratory, Baylor Sammons Cancer Center, Dallas, TX; 4Department of Bioimmunotherapy, M.D. Anderson Cancer Center, Houston, TX.

The mda-7 (IL-24) gene is a novel tumor suppressor with cytokine properties. In an ongoing Phase I dose-escalating clinical trial, mda-7 was administered using a non-replicating adenoviral construct (INGN 241; Ad-mda7). Patients with surgically resectable advanced cancers received single intratumoral injections of 2X1010to 2x1012 viral particles (vp). To date, six cohorts (18 patients) have completed enrollment and two other cohorts have been partially enrolled. INGN 241 appears to be safe and well tolerated with pain at the injection site, transient low grade fever and mild flu-like symptoms being the primary toxicities. To characterize the effects of intratumoral mda-7 treatment, injected lesions were surgically excised at 24-96 h postinjection, serially sectioned, and analyzed for vector DNA and RNA distribution, MDA-7 protein expression, apoptotic activity, as well as iNOS and P-catenin expression. By DNA PCR analysis, INGN 241 copy number ranged from 7x106/ug DNA in low dose treated patients to up to 4x108/ug in patients who received high dose. The highest vector copy number was located at the center of the injected lesion, although vector DNA could routinely be detected in sections up to 1 cm from injection point. mRNA distribution mirrored DNA distribution. By IHC analysis, strong MDA-7 protein expression was found in all injected lesions. Up to 80% of MDA-7 positive cells were found at the center of the high dose-injected tumor, as compared with up to 20% positive staining cells following low dose injection. Non-injected controls were uniformly negative. Further, areas of MDA-7 expression exhibited increased apoptotic activity as defined by TUNEL staining. Apoptosis was most intense in the center of the lesions, with up to 70% of cells being positive; while sections in the periphery also showed a heightened TUNEL reaction compared with uninjected lesions. A marked reduction and/or redistribution of P-catenin expression from the nucleus to the plasma membrane was seen in 8 of 8 INGN 241-treated tumor lesions tested and was consistent with preclinical findings. Markedly reduced iNOS expression was also observed in the limited number of melanoma cases entered into trial. Thus, INGN 241 can induce apoptosis as well as modulate gene expression in a large percentage

of tumor volume. Systemically, vector DNA in plasma varied with treatment dose and time post-injection. Systemic immune responses to INGN 241 were analyzed via serum cytokines and lymphocyte subsets. A majority of patients exhibited transient increases in systemic cytokines (IL-6, 14/18 patients tested; IL-10, 15/18; gIFN, 8/18; TNFa, 10/18). Some high dose patients also exhibited increases in intratumoral expression of IL-6, gIFN and IL-10 cytokine mRNAs. Further, CD3+ CD8+ T cells were increased by 30±13 % at day 15 post-INGN 241-treatment. These findings show that INGN 241 increased systemic TH1 cytokine production and mobilized CD8+ T cells. Immune activation by INGN 241 is likely to augment the direct tumor-selective apoptotic activity of this novel gene therapeutic.

1158. A Phase I/II Dose Escalation Trial of the Intra Prostatic Injection of CG7870, a Prostate Specific Antigen-Dependent Oncolytic Adenovirus in Patients with Locally Recurrent Prostate Cancer Following Definitive Radiotherapy

Theodore DeWeese,1 Elayne Arterbery,2 Jeff Michalski,3 John E. Sylvester,4 Martha K. Terris,5 George Wilding,6 D. C. Yu,7 Laura Moore,7 Lenna Kimball,7 Dale Ando,7 David Kirn.7 'Johns Hopkins Oncology Center, Baltimore, MD; 2Wayne State University, Rochester, MI;3Washington University Medical Center, St. Louis, MO; 4Seattle Prostate Institute, Seattle, WA; 5Stanford University Medical Center & VA Palo Alto Health Care System, Palo Alto, CA; 6University of Wisconsin Comprehensive Cancer Center, Madison, WI; 7Cell Genesys, Inc., Foster City, CA.

CG7870 is a genetically engineered adenovirus with the PSA and Probasin promoter and enhancer elements inserted upstream of the viral E1B and E1A genes, respectively. Control of viral replication by these two prostate-specific regulatory elements has resulted in high specificity and potency against prostate cancer in preclinical xenograft models with CG7870. Twenty patients with locally recurrent hormone naive prostate cancer following definitive radiotherapy or brachytherapy have been enrolled in this phase I/II trial. An intraprostatic injection of CG7870 was administered to each patient as part of a dose-escalation design. Ten patients were treated at a dose level of 1 x 1012 particles (p.u.), 5 patients at 3 x 1012 p.u. and 5 patients at 1 x 1013 p.u.. Treatment was well-tolerated with no related serious adverse events. One NCI-CTC treatment-related grade III adverse event occurred in a patient treated at the 1 x 1013 dose level; the patient had a grade 3 elevated d-dimer associated with grade 2 mild LFT elevations which resolved in 29 days. Patients were evaluable for PSA response if baseline was abnormal (> 5 ng/ mL). PSA decreased by 25-50% in 8 of 12 evaluable patients (67%) and 3 of 3 evaluable patients (100%) at the highest dose level. With a median follow-up of approximately 6 months, 75% of evaluable patients remained progression-free; the median PSA progressionfree survival has not been reached. These data indicate that intraprostatic administration of CG7870 is feasible and is associated with an acceptable toxicity profile. The decrease in PSA levels observed in most evaluable patients to date suggests biologic antitumor activity of CG7870.

Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy