Gene Therapy for Infectious Diseases

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effect. We designed and executed a Phase I, single administration, dose escalation trial of tgAAVCF delivered using the Pari LC Plus™ nebulizer. Four cohorts of 3 patients each were treated with increasing doses of the study agent, beginning with 10E110 DRP [DNase-resistant particles] and escalating in 10-fold increments to 10E113 DRP. Sequential bronchoscopies were performed at timed intervals post-administration to gather samples for molecular analysis of gene transfer and expression, evaluation of cytokine levels, and analysis of neutralizing antibodies. All 12 patients completed the study, including the 90 day follow-up period. No serious adverse events attributed to the study agent were observed. Data on clinical findings, molecular analysis of gene transfer and expression, vector neutralizing antibodies, and cytokine levels in bronchial wash fluid will be presented.

376. Multiple Cycles of Ex Vivo Gene Therapy for X-linked Chronic Granulomatous Disease (CGD) Sustain Production of Oxidase-normal Peripheral Blood Neutrophils

HL Malech*, ME Horwitz*, N Whiting-Theobald*, GF Linton*, JA Miller*, SM Holland*, EJ Read*, SF Leitman*, I Kato†, MR Brown*, TA Fleisher*, DE Van Epps‡ *National Institutes of Health, Bethesda, MD †Takara Shuzo Co., Ltd., Biomedical Group, Otsu, Japan ‡Nexell Therapeutics Inc. Irvine, CA

CGD is an inherited immune deficiency in production of neutrophil superoxide anion and hydrogen peroxide causing recurrent pyogenic infections. About 65% of CGD cases are X-linked (X-CGD) involving mutations in the gp91phox subunit of phagocyte oxidase, and 30% involve the p47phox subunit. In a previous study (Malech et al, PNAS 94:12133, 1997), we reported 5 patients with p47phox-CGD treated with a single infusion of autologous CD341 peripheral blood stem cells (PBSC) transduced with MFGSp47phox murine retrovirus vector without marrow conditioning. Patients produced small numbers of oxidase normal neutrophils peaking at 3-5 weeks at 0.004 to 0.05% of neutrophils and remaining detectable at lower levels for 2-5 months. In the current study we used a similar approach to treat 4 X-CGD patients hospitalized with liver abscess or pneumonia with multiple cycles of ex vivo gene therapy. Special features of this study included: mobilization of PBSC with 8 daily subcutaneous injections of 50 ug/kg flt3L plus 5 ug/kg GMCSF (Mobist™ and Leukinet, Immunex Corp.); selection of CD341 PBSC from apheresis product with the Isolext 300i stem cell collection system (Nexell Therapeutics Inc.); culture and transduction in gas permeable flexible plastic containers (X-Fold™, Nexell) coated with human recombinant fibronectin fragment (RetroNectin™, Takara Shuzo Co.); use of serum free culture medium and vector supernatant (X-VIVO 10™, Biowhittaker Corp.) with flt3L and Pixykine (Immunex) plus SCF as growth factors; and use of high titer MFGSgp91phox vector (.2x10exp7/ml) derived from 293 amphotropic producer. We administered to each patient 2-4 infusions (two or more month intervals) of 2 to 44 x 10exp6/kg autologous transduced CD341 cells (average 70% transduced, range 45-92%). Three of four X-CGD patients demonstrated sustained and continuous production of oxidase normal neutrophils for 6-14 months. As in the previous study, peak levels of oxidase normal neutrophils occurred at 3-8 weeks after each cycle of therapy. However, the improvements in PBSC harvest, selection and transduction resulted in higher peak levels of functionally normal gene-corrected neutrophils (up to 1 of 1% in one patient) and likely contributed to the 4 prolonged effect. Two of three patients producing functionally normal gene corrected neutrophils had large liver abscesses which had been intractable to cure with conventional therapy, and the multiple cycles of gene therapy were administered in this context. After months of conventional therapy plus gene therapy, liver infections in these two patients resolved with patients currently off intravenous antibiotics. In one patient oxidase normal neutrophils were detected in pus obtained from a liver abscess drainage tube, demonstrating normal trafficking of gene corrected neutrophils. In another patient demonstrating production of oxidase normal neutro-

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phils following gene therapy, a bacterial pneumonia resolved while conventional antibiotic therapy was administered. Although the role of the gene therapy in resolving the infections is speculative, it is clear that the gene therapy procedures were well tolerated and did not interfere with resolution of the infections. Based on these studies, it is reasonable to consider studies of the efficacy of gene therapy in the treatment of severe intractable infections in CGD patients.

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377. Potent Inhibition of HIV-1 Infection by Nucleolarlocalized Ribozyme and RNA Decoys Alessandro Michienzi, Laurence Cagnon, Ingrid Bahner, John Rossi Department of Moleclar Biology, Beckman Research Institute of the City of Hope

The HIV regulatory proteins Tat and Rev accumulate in the nucleoli of human cells. However, no functional role has been ascribed to this localization. In addition to its role in ribosomal RNA synthesis and processing as well as ribosomal subunit assembly, the nucleolus is the site of many post-transcriptional modifications. Several cellular messages traffic through the nucleolus, although the functional role of this trafficking has not yet been elucidated. Given that Tat and Rev have nucleolar localization signals, we reasoned that localizing the HIV-1 Rev binding element (RBE) along with TAR and an anti-HIV-1 ribozyme in the nucleolus might enhance the anti-viral efficacies of these RNAs by co-localizing them with the viral proteins or RNAs. To accomplish this, we inserted either the ribozyme or decoys within the apical loop of the small nucleolar RNA U16, and expressed these constructs in HIV-1 infectible cells under the control of the human U6 promoter. Each of the constructs was expressed from the backbone of a retroviral vector, and strong, stable expression patterns were observed. In situ hybridization analyses demonstrated that the chimeric transcripts localized exclusively to the nucleolus. When cells expressing either the U16-rz or U16-RBE were challenged with HIV-1, striking inhibition was observed as measured by p24 antigen production, HIV RNA production and fluorescent immunostaining. For the ribozyme construct, specificity was demonstrated by failure of the mutant ribozyme to inhibit HIV-1 infection. Additionally, the wild type ribozyme did not inhibit HIV-2, which lacks the ribozyme cleavage site. The pronounced inhibitory effects of nucleolar localization strongly support a role for nucleolar trafficking of HIV RNAs during the infectious cycle. At the time of this writing, we have not yet tested nucleolar localized TAR for its antiviral activities, but the results from these analyses will also be reported. From the standpoint of gene therapy for HIV infection, nucleolar localized therapeutics could be extremely effective in blocking viral replication since this is a highly concentrated compartment. In addition to the therapeutic potential of nucleolar localized anti-HIV RNAs, these studies open the possibility of exploiting other targets, both cellular and viral which function in this nucleolar localization process.

378. Gene Delivery to Inhibit HIV-1 using rSV40 Vectors

David S. Strayer*†, Mohamad BouHamdan†‡, Geetha Jayan*†, Sergei Yurasov§¶, Harris Goldstein§¶, JD Chen‡\, Michael Rosenzweig‡\, R. Paul Johnson‡\, Roger J. Pomerantz†‡ *Dept. of Pathology †Thomas Jefferson University ‡Dept. of Medicine §Dept. of Pediatrics ¶ Albert Einstein College of Medicine \Harvard Medical School Effective inhibition of HIV-1 using gene delivery techniques has been observed in cultured HIV-1-susceptible cells, treated with retroviral vectors carrying a variety of therapeutic transgenes, and usually selected to enrich for expressing cells. Transgenes MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

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delivered in this fashion include single chain antibodies, dominant negative mutants of important HIV-1 proteins, ribozymes, decoys, intrakines, and others. Limitations of retroviral gene delivery include relatively low vector concentrations and transduction efficiencies, the need to simulate target cells ex vivo, etc. We devised recombinant viral vectors based on large T antigendeleted SV40 genomes (rSV40) and applied these vectors to the experimental therapy of immunosuppressive lentivirus infection in vitro. The strengths of rSV40 vectors for this purpose include high production titers (up to 10(12) infectious units (IU)/ml), high transduction efficiency (.95%) without selection, ability to transduce both dividing and resting cells permanently and with comparable efficiency, lack of immunogenicity, stable long-term transgene expression, and the ability to transduce the same cells multiple times with rSV40 vectors carrying different transgenes. We have used rSV40 vectors to transduce human cell lines and primary peripheral blood cells, as well as normal human and simian CD341CD38- bone marrow progenitor cells. Transgenes that have been delivered in this setting include single chain antibodies directed against HIV-1 integrase, HIV-1 reverse transcriptase, and the HIV-1 coreceptor, CXCR4; polyTAR decoys; and RevM10, a dominant negative mutant form of HIV-1 Rev protein. In all cases, transduction efficiencies exceeded 50% without selection, and usually exceeded 95%. Transgene expression was stable in culture and following reimplantation in vivo for the duration of these studies (up to 3 months). Delivered by SV40, all of these transgenes protected unselected susceptible cells from challenge with HIV-1 and, where applicable, SIV. Simian CD341 cells were transduced with rSV40 vectors carrying polyTAR decoys without selection and induced to differentiate into CD41 T lymphocytes. These CD41 derivatives of transduced CD341 progenitors were highly resistant to challenge with a pathogenic strain of SIV (SIVmac239). Human fetal thymic tissue implanted into SCID mice was transduced in vivo with rSV40 carrying a single chain antibody vs. HIV-1 integrase, then challenged in vivo with HIV. Substantial protection from the HIV-1 challenge was observed. We have, additionally, found that human cell lines can be transduced in sequence and without selection, with rSV40 vectors carrying different anti-HIV-1 transgenes, resulting in .95% of cells stably expressing both transgenes simultaneously. Taken together, these studies suggest that rSV40 vectors may be important and effective delivery vehicles for the transduction of bone marrow progenitor cells and their progeny‘, particularly with the goal to inhibit infection with immunosuppressive lentiviruses.

the human T cell leukemia virus II envelope protein was then developed. A stable producer cell line was made by transfecting the clone of the pro-HGTV-43 vector DNA into this packaging cell line. Transducing vector produced from this cell line has consistent titers of greater than 107 per ml. A transduction rate of 25-55% of CD341 cells has been demonstrated by colony assay in which HGTV-43-transduced CD341 cells were grown as individual clones to be assayed for antisense RNA expression. Of particular note, the maximum extent of HGTV-43 transduction of CD341 cells was obtained within the 18-hour period immediately after removal of CD341 cells from the HIV-1 infected individual and in the absence of factors used to promote cell division including both stromal cell “feeder” layers, and exogenous cytokines such as IL3, IL6 and SCF. In a clinical trial using this vector, approximately 1010 PBMC were collected by leukapheresis from HIV-1 seropositive individuals post granulocyte stimulating factor (GCSF) treatment. After CD341 cell enrichment on an Isolex 300i column, ;108 PBSC (peripheral blood stem cells) were transduced with HGTV-43 followed by autologous infusion without ablation. Differentiation of transduced CD341 PBSC within HIV-1 seropositive individuals into PBMC was demonstrated by detection of U1/anti-HIV-1 antisense RNA expression in at least one out of every 105 PBMC five months post-infusion. Real-time RT-PCR was performed with cellular RNA isolated from PBMC collected pre-infusion, first week post-infusion, and at monthly intervals thereafter. The identity of U1/anti-HIV-1 antisense RNA-specific RT-PCR amplicon was confirmed by restriction enzyme digestion followed by gel electrophoresis. More importantly, at five months post-infusion, CD41 cell maturation from infused CD341 cells was verified by detection of U1/anti-HIV-1 antisense RNA expression in CD41-enriched cell populations using real-time RT-PCR. Monitoring patients for the presence of this antisense RNA (in both PBMC and CD41enriched PBMC) is ongoing. The efficacy of triple U1/anti-HIV-1 antisense RNA on the inhibition of HIV-1 replication in HIV-1 seropositive individuals is currently under evaluation. These results are a significant and perhaps unique example of the survival of transduced cells within a nonablated adult human subject with continued transgene activity and with replication and differentiation of the transduced CD341 cell.

379. In Vivo Maturation of U1/Anti-HIV-1 Antisense RNA-expressing CD41 Cells from Transduced CD341 PBSC in HIV-1 Seropositive Individuals

There is an acute need for effective technologies to optimize viruses for many clinical applications. Viral vaccines need to be optimized for various properties relating to efficacy, safety and manufacturing issues. Deficiencies in all viral vectors for gene therapy have severely limited their clinical efficacy. Technology for viral optimization is also needed to adapt viruses to grow in model laboratory animals and in tissue culture for use in antiviral drug and vaccine testing (e.g. HIV, Hepatitis B and C). The improved viruses must meet a combination of requirements that the native viruses generally do not possess and this combination of requirements is specific for each application. The absence of an effective technology for optimizing multiple viral properties simultaneously for complex requirements has been a major limitation for engineering viruses. Rational design has occasionally allowed the optimization of single viral properties, but it typically introduces directed mutations and does not broadly search sequence space for sequences that offer the best balance for all the complex requirements. Furthermore, the molecular changes required to obtain desired properties are effectively impossible to predict due to the complex interactions with many other viral and host components. A traditional approach for engineering viruses to obtain specific properties has been to accumulate random point mutations. Viral populations are replicated under selective conditions that favor viruses with point mutations that impart the desired properties. However, phenotypic changes re-

Dakai Liu*, Qi Wu*, Jeffrey Ng*, Joe Ho*, Elizabeth Dunn†, Morton J. Cowan†, Marcus A. Conant†, Barbara E. Thalenfeld*, Dean L. Engelhardt* *Enzo Therapeutics, Inc., Farmingdale, New York †University of California San Francisco, San Francisco, California A murine retrovirus-based transducing vector HGTV-43 was developed to deliver three U1/anti-HIV-1 antisense RNA genes into CD341 cells. This vector is made up of modified MMLV LTR’s and three U1 genes in their entirety, each containing a genetic antisense sequence nested within the RNA-coding portion of the gene. The three genetic antisense sequences are complementary to three separate sequences of the HIV-1 genome including one against the TAR region and two against two separate sites of the TAT region. When U937 cells, a line of human promonocyte origin, were transfected to express these three antisense genes and subsequently challenged with HIV-1 (using both patient isolates and laboratory strains) virus production was completely blocked as measured by p24 production and RT-PCR of the input virus. In addition no integration of the HIV-1 viral DNA was detected. A packaging cell line capable of pseudotyping HGTV-43 with MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

380. Applications of MolecularBreedingtm Technology to Viruses Nay Wei Soong, Willem Stemmer Maxygen Inc., Redwood City, CA.

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NEUROMUSCULAR II quiring complex combinations of mutations are very difficult to obtain by either rational design or by random point mutation, even with many cycles of selective passaging. In contrast to these approaches, breeding is a proven and powerful method for further improving complex and highly functional sequences while balancing many requirements simultaneously. Viruses can be readily bred by a process we have developed, called MolecularBreedingtm technology or DNA shuffling. A major advantage of breeding is that it does not require a large amount of prior information. Related viral strains obtained from natural diversity have evolved separately under different selective pressures and represent a rich source of proven, functional diversity. DNA shuffling gently permutates the natural diversity of these related sequences by homologous recombination, resulting in a library of chimeric variants. The screening or selection of such viral libraries needs to be carefully planned to favor the desired changes while retaining other desirable viral properties. Permutation of functional diversity by shuffling allows the sparse sampling of a much greater area of sequence space. This quickly leads to complex combinations of functional mutations that provide a better balance of the viral properties that are required for a particular clinical application. We present here several applications of molecular breeding of viruses for vector development, DNA vaccines and animal models. Results on the directed evolution of a novel tropism in MLV, improved immunogenicity of a Hepatitis B DNA vaccine and a macaque model for HIV-1 will be discussed.

381. Inhibition of Human Immunodeficiency Virus-1 Replication by a Retroviral Vector Targeting both Viral Entry and Viral Replication Barbara C. Engel*, Gerhard Bauer*, David N. Selander*, David C. Bockstoce*, Si-Yi Chen†, Donald B. Kohn* *Division of Research Immunology/BMT, Childrens Hospital Los Angeles, CA †Baylor College of Medicine, Houston, TX

The intrakine RANTES-KDEL has been shown to efficiently down-regulate the expression of CCR5, the co-receptor for macrophage-tropic HIV-1 strains (Yang et al., PNAS 94, 11567-72; 1997), thus preventing virus entry into a potential target cell. Several approaches have been described to interfere with HIV-1 replication once the virus has entered the target cell. One such strategy involves the overexpression of RRE sequences that act as a decoy and compete with the Rev-binding-element in the HIV-1 transcript for the binding of Rev protein. In an effort to target viral entry and viral replication at the same time, we designed a retroviral vector that combines the intrakine with the RRE-decoy strategy. We constructed a MoMuLV-based vector containing the RANTES-KDEL construct driven by the MoMuLV long terminal repeat (LTR), and an RRE decoy, using eGFP as a marker gene (MND-RANTES-KDEL-IRES-eGFP-RRE). The vector was packaged using the Gibbon Ape Leukemia Virus (GALV) envelope. PM1 cells (human T cell line expressing CCR5) were transduced with the combination vector or a control vector (MND-RANTESKDEL-IRES-eGFP, MND-X-IRES-eGFP) and sorted for eGFP expression. Down-regulation of CCR5 was confirmed by FACS analysis. The cells were then challenged with the macrophage-tropic HIV-strain JR-FL over a range of MOI (0.003 to 0.1). In the negative control cells (transduced with the control vector or untransduced), RT levels started to rise after day 9 of infection and continued to rise until day 21. The cells transduced with the vector containing only the intrakine started to put out increasing amounts of RT at day 15. Meanwhile, the RT acticvity in the cells transduced with the combination vector remained very low to undetectable over the entire period of 21 days. Even at a relatively high MOI of 0.1, the RT values of the protected cells were 10 times lower than those produced by the cells containing the intrakine alone. Our data suggest that the combination of an intrakine with an RRE decoy efficiently suppresses HIV-1 replication in vitro. In ongoing experiments, we are also investigating

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the anti-HIV effects of an MND vector containing the RRE decoy alone. Studies are also underway to assess the potential anti-HIV effects of the combination vector in CD341 cells.

NEUROMUSCULAR II 382. Promoter-based Identification, Selection and Extraction of Neural Stem Cells from the Fetal Human Forebrain

Abner Louissaint, Jr.*, H. Michael Keyoung*, Neeta Singh Roy*, Abdellatif Benraiss*, William K. Rashbaum†, Ayano Kawaguchi‡, Hideyuki Okano‡, Steven A. Goldman* *Dept. of Neurology and Neuroscience, Cornell Univ. Medical College and New York Presbyterian Hospital, New York, NY 10021 †Dept. of Obstetrics and Gynecology, Cornell Univ. Medical College and New York Presbyterian Hospital, New York, NY 10021 ‡Dept. of Neuroanatomy, Osaka Univ. Graduate School of Medicine (CREST), and Japan Science and Technology Corporation, Suita, Osaka, Japan. Neural precursor cells are widespread in the forebrain ventricular lining, and may provide a cellular substrate for brain repair. We have previously isolated and separated committed neuronal precursor cells from fetal animals, by sorting forebrain cells transfected with plasmid DNA encoding the gene for green fluorescent protein (hGFP), driven by the early neuronal promoter Ta1 tubulin (Nature Biotechnol. 16:196-201). We have now extended this approach to include the isolation and purification of neural stem cells from the fetal human brain by sorting on the basis of transcriptional activation of either the nestin enhancer, a regulatory sequence that directs gene expression to neural progenitor cells, or the promoter for musashi-1, a mammalian RNA-binding protein also expressed in neural progenitor cells. Ventricular zone samples were obtained and dissociated from fetuses ranging from 12-23 weeks of gestational age. The resultant monolayer cultures were either transfected with plasmid DNA encoding EGFP under the control of the nestin enhancer (E/nestin:EGFP; Nature Med., 3/2000), or infected with a DE1DE4 type 5 adenoviral construct encoding hGFP regulated by the musashi-1 promoter (AdP/msi:hGFP). Four days later, the cells were re-dissociated, and the GFP expressing cells selected by fluorescence-activated cell sorting (FACS). An average of 2.44 6 0.41% of the cells were gated by FACS on the basis of E/nestin-driven GFP, while 1.23 6 0.21% of these cells were sorted on the basis of musashi promoter-regulated GFP. The majority of E/nestin:EGFPsorted cells rapidly expressed the early neuronal proteins Hu and TuJ1/beta(III)-tubulin; in the week thereafter, most matured to coexpress neuronal MAP-2. Retroviral lineage analysis then revealed that individual E/nestin-sorted cells could give rise to neurons and glia, though neuronal progeny predominated under the culture conditions employed. Clonal analysis was also accomplished by isolating E/nestin:EGFP-sorted cells and replating them in low density/high viscosity methylcellulose culture, which allowed single cells to be observed as such without reaggregation. The individual E/nestin(1) cells thereby observed generated spheres of cells, in which both neurons and astrocytes arose. Similarly, most P/msi: hGFP(1) cells expressed nestin protein, but neither neuronal TuJ1 nor astrocytic GFAP, in the week before FACS. Yet within a week thereafter, most P/msi:hGFP-sorted cells developed either GFAP or TuJ1-immunoreactivity. Limiting dilution with clonal sphere expansion from single AdP/msi:hGFP-sorted cells then confirmed the co-generation of neurons and glia from single P/msi:hGFP(1) cells. Upon xenograft to E17 embryonic rodent forebrain vesicles, the E/nestin:EGFP-sorted cells survived, and by 17 days of postnatal age had developed into all major neural phenotypes in a largely context-dependent fashion. Thus, the use of fluorescent transgenes regulated by early neural promoters, whether delivered via plasmid transfection or adenoviral infection, can permit the identification and enrichment of neural stem cells from the fetal human brain, in a form that allows their heterologous implantation. A. Louissaint, Jr. and M. Keyoung contributed equally to this MOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy