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In light of findings demonstrating that the macaque TRIM5alpha protein inhibits infection of cells by human immunodeficiency virus (HIV)-1, simian immunodeficiency virus (SIV)-based lentiviral vectors may have distinct advantages over HIV-1 vectors for the transduction of macaque hematopoietic stem cells. We evaluated the ability of an SIV vector (VRX859) encoding an antisense SIV envelope sequence and enhanced green fluorescent protein (GFP) to inhibit viral replication and to transduce rhesus CD34(+) lymphoid progenitor cells. After infection with homologous SIV strains, CD4(+) cell lines transduced with VRX859 exhibited more than 600-fold inhibition of viral replication compared with control cells. Less inhibition was observed with the divergent SIV strain SIVsmE660. Partial inhibition of a chimeric simian-human immunodeficiency virus, which contains an HIV-1 envelope in an SIV backbone, was observed, suggesting that the SIV vector also contributes to viral inhibition independent of the antisense envelope inhibitor. Transduction of rhesus CD34(+) cells with VRX859 at various multiplicities of infection resulted in transduction efficiencies comparable to those obtained with the HIV vector VRX494. However, when we evaluated transduction of rhesus T lymphocyte progenitors by examining GFP expression in CD4(+) T cells derived from transduced CD34(+) cells, we observed more efficient transduction with the SIV-based vector. GFP(+)CD4(+) T cells derived from VRX859-transduced CD34(+) cells strongly inhibited SIVmac239 replication as compared with control CD4(+) T cells. The ability of this SIV-based vector to mediate potent inhibition of SIV replication, coupled with its efficient transduction of rhesus hematopoietic progenitor cells, make it an important candidate for proof-of-principle experiments of stem cell gene therapy in the SIV-macaque model.

Meeting abstracts Genetically engineered T cells have broadened the opportunity for use of T cell immunotherapy in cancer patients. The possibility of improving T cell efficacy via introduction of additional genes potentially gives them an advantage over TIL. Studies in adoptive T cell transfer

3043 Background: Studies in adoptive T-cell transfer have suggested that persistence of the transduced T-cells is central to the viability of this therapeutic option. The objectives of this phase I...

The recent development of leukemia in three patients following retroviral vector gene transfer in hematopoietic stem cells, resulting in the death of one patient, has raised safety concerns for the use of integrating gene transfer vectors for human gene therapy. This review discusses these serious adverse events from the perspective of whether restrictions on vector design and vector-modified target cells are warranted at this time. A case is made against presently establishing specific restrictions for vector design and transduced cells; rather, their safety should be ascertained by empiric evaluation in appropriate preclinical models on a case-by-case basis. Such preclinical data, coupled with proper informed patient consent and a risk-benefit ratio analysis, provide the best available prospective evaluation of gene transfer vectors prior to their translation into the clinic.

This review summarises an emerging viral vector system for use in human gene therapy - lentiviral vectors. Lentiviral vectors have several advantages over existing viral vectors. They can stably express transgenes in non-dividing cells in vivo without provoking a significant immune response. They can be produced to a high titre and can be pseudotyped with heterologous envelope proteins to confer broad tropism. Although not without safety concerns, the properties of lentiviral vectors makes them an attractive choice for human gene therapy.

This review is intended to exemplify the roles and responsibilities of the two agencies under the Department of Health and Human Services, the National Institutes of Health and the Food and Drug Administration, that have oversight for human gene transfer clinical protocols, as seen through our experience of bringing a first-in-its-class lentiviral vector to clinical trials. In response to the changing circumstances in gene therapy research between 1999 and 2002, the concerns of these agencies regarding gene therapy have been evolving. This review provides an overview of the major safety concerns regarding insertional oncogenesis, the generation of a replication- competent lentivirus (RCL), and vector mobilization thought to be related to lentiviral vectors, which had to be addressed during the regulatory review process before initiating the clinical trial. Specific monitoring assays to address these concerns were established to test for RCL generation, vector mobilization, persistence of vector-modified cells, and abnormal clonal expansion of modified cells. We hope to provide a basic understanding and appreciation of the regulatory process and major safety concerns, toward providing useful insight to those presently embarking on the development of clinical application of lentiviral vectors.

We report the design of a unique two-plasmid production system for the first lentiviral vector to be evaluated in humans, VRX496. VRX496 is an optimized VSV-G pseudotyped vector derived from HIV-1 that expresses antisense to the HIV envelope gene. We found that a two-plasmid approach to production resulted in higher vector production titers when compared with a three-plasmid approach, which is particularly important for vector production at the large scale. Therefore, we carefully designed a single packaging construct, VIRPAC, for safety by reducing its homology with VRX496 and by insertion of functionally validated genetic elements designed to reduce the risk of generation of a replication-competent lentivirus (RCL). A native cis-acting ribozyme is used to prevent read through into the envelope gene from the upstream gag-pol genes in the packaging vector, thus preventing RNAs containing gag-pol and env together for comparable safety to a three-plasmid system. We demonstrate that there is no significant in vivo vector mobilization using a primary SCID-hu mouse transplantation model, which correlates with the presence of an anti-HIV payload and suggests that inclusion of antisense may be a useful tool to restrict mobilization in other vector constructs. Gene transfer is achieved using a one-step transduction procedure that is simple and clinically translatable, which reaches stable transduction efficiencies of >99% in CD4+ T lymphocytes within 3 days of culture initiation.

Progress in developing effective gene transfer approaches to treat HIV-1 infection has been steady. Many different transgenes have been reported to inhibit HIV-1 in vitro. However, effective translation of such results to clinical practice, or even to animal models of AIDS, has been challenging. Among the reasons for this failure are uncertainty as to the most effective cell population(s) to target, the diffuseness of these target cells in the body, and ineffective or insufficiently durable gene delivery. Better understanding of the HIV-1 replicative cycle, host factors involved in HIV-1 infection, vector biology and application, transgene technology, animal models, and clinical study design have all contributed vastly to planning current and future strategies for application of gene therapeutic approaches to the treatment of AIDS. This review focuses on the newest developments in these areas and provides a strong basis for renewed optimism that gene therapy will have an important role to play in treating people infected with HIV-1.

We have constructed a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector expressing a 937-base antisense sequence against the HIV-1 envelope gene. Transduction of CD4 + T lymphocytes with this vector results in expression of the therapeutic antisense sequence and subsequent inhibition of productive HIV-1 replication. In this report, we examined the effect of antisense-mediated suppression on the potential development of virus escape mutants using a permissive T-cell line cultured under conditions that over serial passages specifically allowed for generation and amplification of mutants selected for by antisense pressure. In the resulting virus clones, we found a significant increase in the number of deletions at the envelope target region (91% compared to 27.5% in wild-type HIV). Deletions were most often greater than 1 kb in length. These data demonstrate for the first time that during antisense-mediated suppression of HIV, mutants develop as a direct result of selective pressure on the HIV genomic RNA. Interestingly, in clones where deletions were not observed, there was a high rate of A-G transitions in mutants at the antisense target region but not outside this region, which is consistent with those mutations that are predicted as a result of antisense-mediated modification of double-stranded RNA by the enzyme double-stranded RNA-specific adenosine deaminase. These clones were not found to be escape mutants, as their replicative ability was severely attenuated, and they did not replicate in the presence of vector.

High gene transfer efficiencies have been difficult to achieve in hematopoietic progenitor cells (HPCs) but are important to therapeutic success of HPC gene therapy. Efficient gene transfer is especially challenging with use of column-purified vector for clinical application, as opposed to centrifuged vector commonly used for research. We investigated novel approaches to increase transduction by using a clinically applicable protocol and quantities of column-purified lentiviral vector. Recognizing the association of adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporters with HPC biology, we investigated the effect of transporter inhibitors on transduction. We found the ABC transporter inhibitor verapamil improved transduction efficiency 2- to 6-fold into CD34+ cells isolated from mobilized peripheral blood, bone marrow, and cord blood. Verapamil also improved transduction in human SCID (severe combined immunodeficient) repopulating cell (SRC) transduction 3- to 4-fold, resulting in 80% to 90% transduction levels in mice receiving primary and secondary transplants without alterations in multilineage reconstitution. Additional ABC transporter substrate inhibitors like quinidine, diltiazem, and ritonavir also enhanced transduction 2- to 3-fold, although ABC transporter inhibitors that are not substrates did not. Enhanced transduction was not observed in mature hematopoietic cells, neurospheres, mesenchymal stem cells, or hepatocytes. Enhancement of transduction in HPCs was observed with vesicular stomatitis virus-G (VSV-G)-pseudotyped lentiviral vector but not with vector pseudotyped with RD114. Thus, we present a new approach for efficient delivery to primitive HPCs by VSV-G-pseudotyped lentiviral vectors. (Blood. 2004;104:364-373)

In our laboratory we have developed lentiviral vectors for use in anti-breast cancer gene therapy. Our specific goal for this project was to evaluate the feasibility of using lentiviral vectors that express anti-HER-2/neu antisense or ribozymes for the treatment of breast cancer. The advantage of lentiviral vectors over other vectors is that lentiviral vectors can transduce human cells with great efficiency. We have demonstrated that HIV- based lentiviral vectors can transduce a variety of human cell types with up to 99% efficiency, as measured by FACS analysis of GFP expression. Specifically, these vectors can transduce human breast cancer cells with up to 90 to 97% efficiency. HER-2 overexpressing breast cancer cell lines, transduced with a lentiviral vector that expresses an anti-HER-2 antisense sequence, exhibit slower growth in vitro compared to non-transduced cells. In addition, breast cancer tumors in nude mice, formed by injection of breast cancer cells transduced with the anti-HER-2 lentiviral vector, tended to grow more slowly than tumors formed by non-transduced breast cancer cells. Modifications and improvements of the current vector system can potentially generate a potent genetic therapy for breast cancer.

INTRODUCTION: To protect normal bone marrow from chemotherapy in glioblastoma patients, we have developed a novel strategy by introducing a strong DNA repair protein, mutant (P140K) of human methylguanine methyltransferase (MGMT), into patients’ CD34+ hematopoietic progenitors (HPC) by lentiviral gene transfer leading to selective expansion of drug-resistant P140K-MGMT CD34+ cells and their myeloid and immune cell progeny. METHODS: To achieve long-term stable expression of the P140K-MGMT gene, we used a lentiviral vector which integrates into the host genome. However, viral insertion mutagenesis has raised safety concerns; as the previous γ-retroviral vectors were associated with insertion mutations leading to development of acute lymphoblastic leukemia in 20% of treated patients. Nevertheless, new improved lentiviral vector with safe feature of insertion site far away from gene transcription start site has been developed. Here we evaluated the safety of a lentivirus vector under selection pressure of chemotherapy. HYPOTHESIS: Our lentiviral vector is safer than traditional γ-retroviral vectors as evident by lack of early clonal dominance even with a chemo-selection. RESULTS: Three glioblastoma patients were recruited and underwent resection, after which CD34+ HPC were mobilized with filgrastim, isolated by magnetic bead separation (Miltneyi CliniMACS), and transduced ex vivo with a GMP-grade lentiviral P140K-MGMT vector (Lentigen Corp). Subsequently, patients received radiation/temozolomide for 6 weeks and up to five cycles of monthly O6-benzylguanine/temozolomide (BG/TMZ) treatment. As a result, all three patients demonstrated a 5-15 fold selective expansion of P140K-MGMT positive HPC and their progeny granulocyte and mononuclear cells in peripheral blood and a small number of CFUs from bone marrow indicating a drug-selection mechanism. The viral insertion sites in the cells of these three patients were closely monitored in each chemotherapy cycle and the patients were followed for up to 1 year after the last therapy. We mapped a total of 17,308 viral insertion sites (VIS), for patient 1(6,146), patient 2(2,081) and patient 3(9,081) and the unique viral insertion sites (UVIS) was 382, i.e. 135, 76 and 171 for patient 1, patient 2 and patient 3 respectively. Overall, during the drug-treatment period, there were no persistent UVIS. Moreover, at the conclusion of BG/TMZ treatment, the VIS number sharply diminished. CONCLUSION: Gene transfer of LV MGMTP140K vector into hematopoietic progenitor cells did not lead to clonal dominance during or after drug selection. Dose escalation of BG/TMZ will define hematopoietic tolerance and treatment response. Disclosures Embree: Lentigen Technology Inc: Employment. Dropulic:Lentigen Technology Inc: Employment, Patents & Royalties.

Top of pageAbstract Emerging drug resistance raises concerns about HAART as a long-term therapy. We have developed an HIV-1-based lentiviral vector expressing a 937-base antisense gene against the HIV envelope for autologous T cell therapy for HIV/AIDS. The vector introduces no new sequences to an HIV-infected individual. In preclinical studies, HIV replication was inhibited over 95%, and up to 4 logs, in primary CD4 T lymphocytes isolated from HIV-infected patients. For evaluation of safety and tolerability of this therapy, a Phase I open-label non-randomized clinical trial has been carried out. Five patients who have failed at least two HAART regimens due to intolerance or resistance, and have CD4 counts above 150 cells/mm3 and a viral load greater than 5000 copies/ml, have been serially enrolled for a 6-month study with 21 day, 3, 6, 9, and 12-month monitoring points. Each patient was given a single i.v. dose of |[sim]| 1 |[times]| 1010 genetically-modified autologous T cells, and then monitored for viral load, CD4 count, replication competent lentivirus (RCL), outgrowth of vector-modified cells, immunological parameters, and adverse events. Serious adverse events (SAE) were defined in part by a sustained 30% increase in viral load, or sustained 0.5 log decrease in CD4 count within 3 weeks post dose. All five patients have been dosed, and have completed the critical 3-week safety timepoint. No SAEs related to the product have occurred, and all RCL tests have been negative, including a biological RCL assay performed on apheresed patient PBMCs at 6 months. Patients 1-3 have reached their 1 year follow-up and all three have demonstrated clinically significant (>0.5 log) reductions in their viral loads at 1 year at 0.78 log, 1.61 log, and 0.57 log, respectively. Furthermore, patients 2 and 3 have had a 20% and 30% increase in CD4 counts, respectively, while patient 1 has maintained a steady CD4 count. Patient 4 will reach his 9-month follow up in March, and Patient 5 has just completed his 3-month follow up. Circulating vector-modified cells was detected in patient 2 at one year, to month 9 in patients 1 and 3, and they remain detectable in patients 4 and 5. To date, 211 total integration sites from the transduced clinical products of all 5 patients have been analyzed; initial results indicate that VRX496 preferentially integrates into transcription units and disfavors integration into alphoid repeats, a pattern that is comparable to and indistinguishable from wild type HIV. These data support the safety and feasibility of lentiviral vector therapy in the setting of ex vivo cellular therapy.

We report the testing of an HIV-1-based lentiviral vector expressed in CD4 T cells in a Phase I, open-label, non-randomized gene transfer trial. In this study, 5 patients were each given a single IV dose of approximately 1 |[times]| 1010 autologous CD4 T cells that had been transduced ex vivo with the lentiviral vector VRX496. VRX496 expresses a 937 nt antisense gene against the HIV envelope. All patients had chronic HIV, with CD4 counts above 150 cells/mm3 and a viral load > 5000 copies per ml. The patients had all failed at least two HAART regimens due to intolerance or resistance. All five patients have been dosed, and no serious treatment related adverse events have occurred. Viral loads are shown in the figure; one subject maintained a nearly 2-log decrease in viral load at the 1 year time point. CD4 counts remained stable in all patients and circulating VRX496-modified cells were detected in all patients for at least 6 months. Immune reconstitution is suggested by ELISPOT analysis of PBMC. 4 of 5 patients have increased HIV gag and env specific IFN-g expressing CD8 T cells (702 mean|[plusmn]|269 sem SFU per 1|[times]|106 PBMC at baseline, and 2610|[plusmn]|662 SFU at 3 to 12 months post infusion, P

In light of recent findings demonstrating that the rhesus TRIM-5a protein specifically binds to HIV-1 Gag and blocks the uncoating of viral particles, SIV-based lentiviral vectors have been proposed as a more appropriate vector than HIV-1 based vectors for the transduction of rhesus macaque hematopoietic stem cells. We evaluated the ability of a SIVmac239-based lentiviral vector (VRX859), which encodes 1275 bp antisense SIV envelope and eGFP transcriptionally regulated from the SIV LTR, to inhibit viral replication in vitro and compared transduction of rhesus CD34+ lymphoid progenitor cells with an HIV-1 based lentiviral vector. Initially, we evaluated inhibition by VRX859 of the homologous SIVmac239; the related SIVmac251; the heterologous SIVsmE660 (|[sim]|80% homologous in env); the chimeric SHIV 89.6P and HIV-1 NL4-3. CEMx174 cells, which support replication of both HIV-1 and SIV isolates, were transduced with VRX859 at MOI of 10 and the eGFP sorted population contained 1 proviral copy per cell. Following infection with SIVmac239 and SIVmac251, CEMx174-VRX859 cells exhibited over 1000- and 650-fold inhibition of viral replication compared to control cells. Inhibition of SIVsmE660 and SHIV 89.6P was weaker, ranging from 3 to 9-fold and 18 to 32-fold. HIV-1 NL4-3 replication in CEMx174-VRX859 cells was similar to control cells. To evaluate transduction of rhesus CD34+ cells and lymphoid progenitor cells, we prestimulated CD34+ cells with Tpo, Flt3L and SCF (50 ng/ml each) for 1 day and exposed cells to VRX859 vector (or the analogous HIV-1 based lentiviral vector VRX494) for 1, 2 or 3 days on Retronectin-coated plates. Transduction of rhesus CD34+ cells with the SIV vector VRX859 over a range of MOIs (3|[ndash]|50 IU per cell) resulted in similar percentages of eGFP expression as observed with the HIV vector VRX494 at similar MOIs. Transduced CD34+ cells were then cultured with rhesus fetal thymus stromal cells to induce T cell differentiation and analyzed by flow for expression of eGFP. eGFP expression in CD4+ T cells derived from CD34+ cells transduced with VRX859 at low MOIs (3|[ndash]|10 IU per cell) was increased from 13- to 2.2- fold compared cells transduced with VRX494. eGFP expression in CD4+ T cells derived from CD34+ cells transduced at higher MOIs (30 and 50 IU per cell) were basically equivalent between VRX859 and VRX494. Rhesus CD4+ T cells expressing eGFP were obtained by cell sorting and expanded with ConA, IL-2 and irradiated human feeders. GFP+ CD4+ T cells derived from VRX859-transduced cells strongly inhibited SIVmac239, SIVsmE660 and SHIV 89.6P replication (up to 1000-fold) as compared to control CD4+ T cells. These results demonstrate superior transduction of rhesus lymphoid progenitor cells with a SIV-based lentiviral vector, particularly at low MOI, and strong inhibition of viral replication, based partially on competition with genomic RNA, in CD4+ T cells derived from transduced CD34+ cells and in a CD4+ cell line. These results support using a SIV-based lentiviral vector in the rhesus macaque model as a valuable preclinical model for stem cell gene therapy for AIDS.

Meeting abstracts Previous studies in adoptive T-cell transfer have suggested that persistence of the transduced T-cells is central to making this therapy a viable option. Understanding the behavior of tumor-reactive T-cells in cancer patients and measuring persistence are two objectives in a phase

TPS1616 Background: The administration of radiation therapy (RT) and concomitant and adjuvant temozolomide (TMZ) for the treatment of newly-diagnosed glioblastoma (GBM) is associated with modestly prolonged survival. However, the benefit of concurrent therapy is restricted to a subgroup of patients whose tumor exhibits methylation of the promoter for methylguanine-DNA-methyltransferase (MGMT). Promoter methylation silences the expression of DNA alkyltransferase (AGT), the product of the MGMT gene which functions to repair alkylated DNA. Unfortunately, the majority of GBMs have non-methylated MGMT promoters and do not appear to benefit from adding TMZ to radiotherapy. Two potential strategies for targeting MGMT-induced chemoprotection in gliomas include depletion of tumor MGMT, and dose escalation of TMZ accomplished by minimizing TMZ-induced bone marrow toxicity. In this study, we propose to combine the irreversible MGMT inhibitor O6-benzylguanine (BG) with infusion of autologous hematopoetic stem cells (HSCs) transduced with lentiviral P140K-MGMT, an MGMT mutant which is highly resistant to inactivation by BG, with the intent to reduce or prevent the myelosuppressive effects of TMZ. The primary objective is to assess the safety and feasibility of combining these two strategies in patients with newly-diagnosed GBM. Methods: This is a three cohort, Phase I study for patients with newly resected supratentorial GBM. There will be at least four evaluable patients per cohort. Cohort 1 will receive standard RT and concomitant TMZ prior to infusion of autologous lentiviral P140K-MGMT transduced HSCs, followed by adjuvant TMZ. Cohort 2 will receive an induction dose of TMZ+BG followed by infusion of autologous LV-P140K-MGMT HSCs prior to concomitant RT +TMZ and adjuvant TMZ. Cohort 3 will include intra-patient dose escalation of TMZ in patients who exhibit detectable levels of P140K-MGMT marked cells in vivo, using the design (i.e. cohort 1 or 2) proven to be safest and most effective. The study is open and has accrued the first patient.

We have assessed the sensitivity of different sites in HIV-1 genomic RNA to ribozymes. Ribozymes targeted to sequences in U5, Pol, Env, RRE, or R were positioned into nef of an infectious HIV-1 provirus. When these proviral DNAs were introduced into HeLa CD4+ cells, recombinant viruses that contain ribozymes tethered to genomic RNA or viral mRNAs were produced. The growth kinetics of ribozyme-containing viruses in CD4+ lymphocytes (MT4 cells) were distinctly delayed when compared to control viruses. On the basis of the ability of a particular ribozyme to inhibit virus replication, we inferred intracellular ribozyme-sensitive sites. We found that although ribozyme sensitivity in vitro could be correlated with predicted secondary structures of target RNAs, such in vivo correlations could not be made when using the HIV provirus model. We conclude that both Zuker algorithm computer modeling of substrate RNA secondary structures and in vitro cleavage efficiencies cannot be reliably used to determine HIV-1 ribozyme sensitive sites in vivo.

Publisher Summary This chapter presents some of the different ways in which RNAs and their cognate binding proteins function to regulate gene activity and discusses potential pharmacological applications of RNAs as gene-modulating agents. The chapter focuses on the use of RNA analogs-oligodeoxynucleotides (ODNs)-in regulating gene expression. It has been suggested that RNA, not DNA, is the ancestral genetic material. Accordingly, it is not surprising that RNA sequences and RNA-binding proteins play important regulatory roles in cellular (and viral) metabolism. The chapter reviews some natural instances of RNA-mediated regulation. The chapter presents examples that provide some guiding principles on how to use RNAs and RNA analogs to subvert unwanted gene expression. The theories supporting use of antisense RNA (DNA), ribozymes, and decoys are sound. The logistics governing specific delivery into appropriate cells, intracellular targeting, high levels of long-term functional expression and minimization of toxicity remain obstacles that prevent useful pharmacological applications.

Abstract 4177 Temozolomide (TMZ) is a standard of care treatment for patients with glioma post surgical resection, but induces dose limiting myelosuppresion. Gene transfer of O6-benzylguanine (BG) resistant mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K) into hematopoietic stem cells (HSCs) protects hematopoiesis from alkylating agents and allows efficient in vivo selection of transduced HSCs in mice, dogs and primates. We designed a clinical trial to improve HSC tolerance of TMZ in patients with glioma [clinicaltrials.gov]. This trial uses a lentiviral backbone for gene delivery, and infuses cells during the active phase of treatment to maximize the potential for stem cell protection and selection. The viral construct was synthesized by Lentigen, with an EF1-alpha promoter driving MGMTP140K (LV-P140K MGMT). Clinical grade vector was subject to safety and toxicity analysis at the request of both NCI and FDA. Transduction conditions were optimized towards a high rate of gene transfer and expression with ≤2 copies per cell to minimize adverse insertions. This study presents the validation of the vector by assessing the engraftment, drug resistance and copy number after gene transfer into human CD34+ cells followed by in vivo selection using a nonobese diabetic (NOD)/severe combined immunodeficient (SCID) gammaC (NSG) mice. We are currently analyzing the insertion sites of the clinical grade LV-P140K MGMT in human cord blood progenitors as a safety monitoring tool for LV-HSC gene therapy. Engraftment of LV-P140K MGMT (LG631) transduced CD34+ cells in irradiated NSG mice, and the analysis of engrafted human CD45+ cells in peripheral blood, spleen and bone marrow showed consistent but slightly decreased (10%) engraftment of transduced cells compared to the freshly isolated non transduced cells and was multilineage. There was no toxicity to the mice or human HSC as a result of transduction with lentiviral vector. The NSG recipients of the LG631 transduced CD34+ cells received two rounds of 3-consecutive day treatments of 30 mg/kg of BG and 60 mg/kg of TMZ, and three weeks after the second round of treatment, expression of MGMT was examined by PCR in the CFUs from the BM of the recipient mice. Combined treatment with BG and TMZ showed efficient selection of human cells and human CFU expressing MGMT (100% after treatment vs 44+/−4.1% without treatment). Copy number and characterization of insertion sites of the clinical grade LV-P140K MGMT in human CD34 cells serve as a critical safety monitoring tool for LV-HSC gene therapy. Insertion site analysis showed a polyclonal pattern and selection did not result in oligoclonality. The insertion database will continue to be made publically available as the data is generated prior to and during clinical trial application. We have fully characterized the preclinical safety and efficacy of a clinical grade lentiviral vector for clinical trial application in patients with glioma. Disclosures: No relevant conflicts of interest to declare.

The activity of a promoter is influenced by chromosomal and cell cycle/replication context. We analyzed the influences of integration and replication on transcription of the human immunodeficiency virus (HIV)-1 long terminal repeat (LTR). We found that one requirement for Tat trans-activated expression differed for integrated versus unintegrated HIV-1 LTR. Specifically, the second coding exon of Tat, previously regarded as functionally dispensable, plays a role in the optimal activation of integrated LTRs. We also found that the transcription profile produced from the HIV-1 LTR is influenced by replication. Autonomously replicating vectors that contain the HIV-1 LTR produced patterns of transcription different from counterpart vectors that do not replicate. Both replicating and nonreplicating HIV-1 LTRs remained responsive to Tat.

The increased cost of goods associated with low-level expression of factor VIII (fVIII) is considered a significant factor in the pricing of recombinant fVIII products. Worldwide treatment of hemophilia A is limited due to the cost of fVIII products, which easily can exceed $100,000 per year. Using state-of-the-art commercial production facilities and technology, recombinant human (h)-fVIII expresses at 100 – 1000-fold lower levels than other plasma proteins. We have developed a novel recombinant fVIII product that overcomes the primary limitation to the manufacture of currently available hemophilia A pharmaceuticals. There are two components to this product: a humanized highexpression hybrid-human/porcine (HP) fVIII transgene, designated ET-801i, and the LENTIMAX™ lentiviral production system. The ET-801i transgene contains elements of porcine fVIII (p-fVIII) sequence in the A1 and A3 domains that facilitate more efficient secretion from production cell lines and is 90% identical in amino acid sequence to h-fVIII. Additionally, lentiviral introduction of the transgene allows achievement of an optimal number of transgene copies and the lentiviral vectors typically integrate into sites of active chromatin, thus facilitating high-levels of transgene transcription. In the current study, we demonstrated that high-expression porcine sequence elements enable 20 – 100- fold superior expression over any previously described fVIII transgene variant. Using a stable baby hamster kidney cell-derived expression system, ET-801i was expressed at a level indistinguishable from p-fVIII, which both were significantly greater than h-fVIII (

We established the first clinical ex vivo HIV-based vector gene therapy trial in humans with HIV+ CD4+ T-cells. Briefly, this therapy involves modifying patient CD4+ T-cells with our modified lentiviral vector carrying an anti-HIV payload. These cells are then activated and expanded, and re-infused back into the patient. However, cGMP regulations require the use of costly clinical grade reagents (i.e. Retronectin™, CD3/CD28 stimulating paramagnetic beads). In an attempt to reduce ex-vivo processing costs, but not at the expense of transduction levels, we sought to determine a way to directly activate CD4+ T-cells with modified lentiviral vectors. 293FT HEK cell lines, used for producing our lentiviral vectors, were modified to co-express the natural CD28 stimulatory ligand B7.2 (CD86) and ICAM-1 (CD54) proteins on their membrane for co-stimulation and anchoring purposes. When CliniMACS purified normal donor CD4+ T cells were co-cultured with CD54/CD86-expressing cells, in the presence of soluble OKT3 CD3 antibody, CD25 and CD69 activation markers were upregulated, indicating that functional proteins were being expressed at the cell membrane. These CD54 and/or CD86 expressing cells could subsequently be transfected with lentiviral vector plasmid constructs in order to produce host-derived CD54 and/or CD86 bearing HIV-based vectors. EGFP-expressing lentiviral vectors, VRX494, with CD54/CD86-modified envelopes were produced both in these cell lines and by transient transfection of all relevant plasmids, and titers were assayed on Hela-Tat cells by FACS. CD54 modified lentiviral vectors showed increased binding to CD4+ T-cells, as evidenced by significant cell clumping. CD86 (as well as CD54 plus CD86) modified lentiviral vector, with soluble OKT3 CD3 antibody, was shown to activate T-cells, above the levels seen with unmodified lentiviral vectors, as evidenced by the increase in cell surface CD25 and CD69 expression and also the increase in cell size. Cellular expansion of modified lentiviral vector transduced CD4+ T cells reached levels close to CD3:CD28 bead stimulated CD4+ T cell controls over a period of 2 to 3 weeks. The CD3/TCR repertoire was assessed by flow cytometry and, compared to the well-established CD3/CD28 coated M450 Dynabeads stimulatory system as a control, no skewing of the repertoire was observed. CD86 was shown to improve levels of transduction in pre-activated lymphocytes with CD3/CD28 coated M450 Dynabeads. However, CD86 co-expression was crucial for transducing minimally activated CD4+ T cells with only soluble OKT3 CD3 antibody. Levels of transduction and activation were on average 2 to 3 times higher with the modified lentiviral vectors. To our knowledge, we are reporting the first generation of lentiviral particles exhibiting an adhesion property with stimulatory abilities. The development of such a lentiviral vector has valuable implications for clinical application by reducing the number of exogenous reagents in large scale cell processing.

Lentiviral vectors (LV) offer improved therapeutic benefit of gene therapy applications as a result of their ability to stably transduce a wide range of cycling and non-cycling cells. The first Phase I clinical trial using a lentiviral vector, VRX496, was initiated in January of 2003. The vector evaluated is an HIV-1 based lentiviral vector, fully gutted but retaining the 5′ and 3′ long terminal repeats, the packaging sequence, cPPT/CTS, splice donor and splice acceptor, and rev response element (RRE). VRX496 contains a splice-independent 937-base antisense sequence targeting the envelope gene in the HIV genome, and a small 186-base tag derived from the GFP gene to serve as a molecular marker for vector in HIV infected patient cells. Five (5) HIV-infected patients with CD4 counts between 200 and 500, and viral loads above 5000 copies/ml, no history of opportunistic infections, who have failed two regiments of highly active antiretroviral therapy (HAART), were serially enrolled in the trial. After leukopheresis, patient’s CD4 T lymphocytes were isolated by negative selection of CD8+ cells, and then cultured for three days in the presence of immobilized CD3/28 beads, and VRX496. Thereafter, the vector and beads were removed, cells were expanded to the dose of 1 x 1010, and then given intravenously over 15 minutes. At the time of this abstract, 4 patients have been dosed and have passed the initial 21-day safety assessment; a fifth patient is scheduled for dosing. No serious adverse events have occurred. All patients have steady CD4 counts, and viral loads have decreased below baseline in all patients, notably from 218,000 pre dose to 36,000 1 year post dosing in the first patient, and from 22,000 pre dose to 1,625 at 9 months post dosing in the second patient; patients 3 and 4 are at 3 months and 21 days post-infusion, respectively. Importantly, persistence of vector-modified CD4 cells are detected in the peripheral blood out to 9 months post dosing. This trial establishes for the first time the safety of lentiviral vectors for clinical gene therapy application.

Recently, we initiated the first ex vivo HIV-based gene therapy trial in humans with HIV+ CD4+ T cells. In this protocol, a modified lentiviral vector carrying an anti-HIV payload is used to modify CD4+ T cells isolated from HIV-infected patients by apheresis and CD8 negative selection. The T cells are activated in the presence of vector and expanded using immobilized CD3/CD28 antibodies, and then infused back into the patient. T cell receptor (TCR) repertoire analysis has value for safety monitoring of adoptive T cell transfers in the detection of aberrant clonal expansions or deletions. In this study, the TCR Vβ repertoire was assessed using a flow cytometry based assay at various time points in the selection/transduction/expansion process of CD4+ T cells. PBMC isolated from whole blood of HIV+ patients were CD4-selected using a CD8 negative selection, followed by enrichment by CD3 antibody. CD4+ purified cells were transduced with the lentiviral vector, VRX496, in the presence of retronectin, and then co-cultured with CD3/CD28 coated M450 Dynabeads for ten days. The TCR Vβ repertoire was assessed in throughout the process using a FACS-based assay that employs a panel of 20 monoclonal antibodies recognizing most of the 24 Vβ families in PBMC and CD4+ T cells. Repertoires from subjects with normal polyclonal TCR profiles were conserved, as shown by the absence of any significant change in any Vβ family. Moreover, the transduction/expansion of CD4+ T cells from a patient with a previously skewed TCR profile allowed the improvement of the TCR Vβ repertoire. Finally, no significant difference was observed in the repertoire of cells transduced with VRX496 versus mock-transduced cells. These data demonstrate stability of the repertoire diversity and thus provide important support information in favor of the safety of a gene therapy approach involving lentiviral vector mediated modification and expansion of CD4+ T-cells.

Lentivirus-based vectors have been considered to offer increased safety in terms of oncogenesis in part since contrary to oncoretroviruses, lentiviruses are not associated with oncogenesis. In support of this, leukemia is not a recognized side effect of HIV infection, even though memory T cells are known to harbor virus for years; additionally, approximately 1 out of every three proviruses are replication incompetent, and therefore cannot kill the host cell, yet these cells also do not turn leukemic. Until recently, retrovirus-mediated oncogenesis remained a theoretical risk, until the two children in the French X-linked SCID trial developed leukemia. Although the development of leukemia may have as much to do with the type of cells used in gene therapy as the vector used, the unfortunate event brought to the forefront the need to determine the specific relative safety of different retroviral vectors. In order to determine the relative safety of our HIV-1-based lentiviral vector to the MLV vector, we are investigating the enhancer activity of the HIV LTR in comparison to the MLV LTR and an enhancer negative promoter control. Preliminary studies in NIH 3T3 cells indicate that the MLV LTR contains an enhancer effect greater than that of the HIV LTR. Currently, studies are being conducted in the CD34+ cells isolated from one of the two children that developed leukemia in the X-linked SCID trial, in order to test the relative enhancer activities in the cellular environment where oncogenesis was first observed in vivo. In parallel, we used LAM-PCR to determine the insertion sites of an HIV-1 based lentivirus vector in several HIV+ patients undergoing evaluation in the first lentiviral vector clinical trial. We compared the distribution of the insertions among the chromosomes to those published in the Bushman publication of 2003; we found a similar pattern of random distribution among all chromosomes except chromosome 18, which appears to be partially refractory to integration. Details of the insertion sites will be presented including the specific location of the insertion within the chromosome, and proximity to genes with oncogenic potential.

Despite the significant advancement in HIV/AIDS treatment with highly active antiretroviral therapy (HAART), HIV/AIDS remains incurable. Limitations of HAART include expense, disabling complications, and a stringent requirement for compliance. In the United States, where HAART has become the standard of care, approximately 20% of newly transmitted virus is already resistant to at least one antiretroviral drug. In anticipation of expanded HAART in Africa, there is concern regarding rapid emergence of drug resistant strains of HIV that brings into question HAART as a long-term solution. We have developed an alternative therapy for HIV/AIDS therapy using an HIV-1-based lentiviral vector expressing a 937-base antisense gene against the HIV envelope, VRX496, for autologous T cell therapy. T cells are transduced with vector in the presence of co-stimulatory CD3/CD28 beads, and subsequently expanded in culture. Preclinical studies demonstrated the feasibility of this approach in normal CD4+ T cells and inCD4+ T cells isolated from 20 early and late stage patients, as defined by viral loads. An efficient gene transfer procedure was established, which demonstrated stable transfer rates of 94% +/- 5%. Antisense inhibited HIV in cultures by >93% over mock transduced controls, regardless of patient status or the tropism of the infecting virus. A Phase I open label non-randomized clinical trial was then initiated to investigate the safety and tolerability of autologous T cells modified with VRX496. In this study, 5 HAART resistant patients having CD4 T cells counts between 200-500 cells/mm3 are serially enrolled in the study that includes 3-week, and 3 and 6-month monitoring points. Each patient is given approximately 1 1010 modified autologous CD4 T cells in a single dose. Patients are monitored for viral load, CD4 count, emergence of any replication competent lentivirus (RCL), immunological parameters, and clinical indications. Adverse events are defined in part by a sustained 0.5 log increase in viral load, or sustained 0.5 log decrease in CD4 count within 3 weeks post dose. To date, 3 subjects have been infused, and the first and second subjects have completed the 6 and 3-month monitoring points, respectively. No adverse events have been noted, and all RCL assays have been negative. CD4 counts remained steady and engraftment of VRX496-modified cells was observed for at least 6 months in the first patient and 3 months in the second patient. At dosing, subject 1 had an average viral load of 188,500 and subject 2 had an average viral load of 40,428. After three and six months post dosing, subjects 1 and 2 respectively, had viral loads below baseline. This interim analysis supports the promise of lentiviral vectors for late stage HIV infection, and that VRX496 could become a viable treatment modality for individuals infected with HIV/AIDS.

Background CD19 redirected chimeric antigen receptor (CAR) T-cell therapy has proven efficacy in relapsed or chemotherapy-refractory (r/r) aggressive B-cell non-Hodgkin lymphoma (B-NHL). However, targeting a single B-cell antigen leads to selective pressure with potential antigen-escape and subsequent relapse. A tandem CAR targeting CD20 and CD19 (pLTG1497) has been developed to overcome this limitation. Preclinical evaluation showed improved anti-lymphoma activity. Thus, we initiated a first-in-human, phase I clinical study of autologous pLTG1497-transduced CAR T-cells (MB-CART2019.1) in r/r B-NHL patients. Aims In this phase I prospective multi-center trial (NCT03870945) we aimed to evaluate the maximum tolerated dose (MTD) of MB-CART2019.1 in adult patients with CD20 and CD19 positive r/r B-NHL as determined by dose limiting toxicities (DLTs). Methods This was a 6+3 trial design with two predefined dose levels (DL1 1x106 and DL2 2.5x106 CAR T-cells/kg body weight, respectively). Secondary endpoints included adverse events (AEs) and best overall response rate (ORR). Pharmacodynamic assessments included maximum concentration (Cmax) of CAR T-cells, time to peak expansion (tmax), AUC (d0 to d28), and persistence. MB-CART2019.1 was produced by lentiviral transduction of autologous fresh leukapheresis in the closed automated CliniMACS Prodigy® System (Miltenyi Biotec, Bergisch Gladbach, Germany). Re-infusion (Day 0) of fresh MB-CART2019.1 was scheduled 14 days after leukapheresis. Fludarabine/cyclophosphamide lymphodepleting chemotherapy was administered from day -5 to -3. Results A total of 12 patients, 6 per dose level have been enrolled and treated between February and December 2019, 5 female and 7 male patients. Median age was 72 y (range 20, 78 y), with 10 patients >65 y and 8 >70 y. Histologies included aggressive B-NHL (11) and mantle cell lymphoma (1). Five (5) patients had refractory disease at study entry and IPI was ≥3 in 7 patients. Median time from leukapheresis to re-infusion was 14 d (range 13, 14 d). No DLT and no cytokine release syndrome (CRS) or neurotoxicity grade ≥3 were observed. One patient in dose level 1 experienced a grade 5 AE, which was due to disease progression. CRS grade 1 occurred in 3/6 patients on DL1 and DL2 each, and CRS grade 2 in 2 patients on DL2. Tocilizumab was given in 1 patient. Neurotoxicity grade 1 occurred in 1 patient on DL2. The above described CRS and neurotoxicity resolved completely. Mean Cmax of MB-CART2019.1 was 348.3 cells/µl (range 3.9, 830.4 cells/µl) on DL1 and 692 cells/µl (range 5.3, 3147.8 cells/µl) on DL2. Mean tmax was 15.8 d (range 9, 21 d) on DL1 and 11.5 d (range 9, 14 d) on DL2. Mean AUC was 3155 d*cells/µl (DL1) and 4339 d*cells/µl (DL2). Persistence of MB-CART2019.1 was observed in 12/12 patients until data cut-off. Altogether 9/12 patients (ORR 75%) responded to MB-CART2019.1 with 5/12 CRs. In DL1 3/6 patients responded (ORR 50%) and in DL2 6/6 patients (ORR 100%). The 3 patients without response to MB-CART2019.1 had a mean AUC0-28 of 870 d*cells/µl, whereas mean AUC0-28 in 9 responders was 4843 d*cells/µl reflecting the correlation between the pharmacodynamic parameters and the clinical response. Responses are ongoing in 5/9 patients, with a maximum duration of response of 330 days at data cut-off. Summary/Conclusions In this first-in-human dose finding study of MB-CART2019.1 no DLT and no severe (grade ≥3) CRS or neurotoxicity were observed. Feasibility and safety were very good in this cohort of elderly r/r B-NHL patients. The sustained expansion of tandem CAR T-cells was accompanied by efficacy: all patients (6/6) treated on DL2 responded and all 5 patients with CR (5/5) are in ongoing remission by the time of this report. Based on the promising risk-to-benefit ratio observed in our study, evaluation of MB-CART2019.1 at a dose of 2.5x106/kg body weight in clinical phase II and phase III trials for patients with relapsed aggressive B-NHL is underway. Disclosures Borchmann: Miltenyi Biotec B.V. & Co. KG: Honoraria. Balke-Want:Miltenyi Biotec B.V. & Co. KG: Honoraria. Ayuk:Celgene: Consultancy, Honoraria; Kite/Gilead: Honoraria; Therakos/Mallinckrodt: Honoraria, Research Funding; Neovii: Research Funding; Novartis: Honoraria. Holtkamp:Miltenyi Biomedicine GmbH: Current Employment. Preussner:Miltenyi Biomedicine GmbH: Ended employment in the past 24 months. Zadoyan:Miltenyi Biomedicine GmbH: Current Employment. Hanssens:Miltenyi Biomedicine GmbH: Current Employment. Kaiser:Miltenyi Biotec B.V. & Co. KG: Current Employment. Jurk:Miltenyi Biotec B.V. & Co. KG: Current Employment. Bürger:Miltenyi Biotec B.V. & Co. KG: Current Employment. Schneider:Lentigen Technology Inc., A Miltenyi Company: Current Employment, Patents & Royalties. Dropulic:Lentigen Technology Inc., A Miltenyi Company: Current Employment. Overstijns:Miltenyi Biomedicine GmbH: Current Employment, Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec B.V. & Co. KG: Current Employment, Membership on an entity's Board of Directors or advisory committees. Scheid:Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; BMS: Honoraria; Amgen: Honoraria; Takeda: Honoraria, Research Funding. Holtick:Miltenyi Biotec B.V. & Co. KG: Honoraria. Miltenyi:Miltenyi Biomedicine GmbH: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Lentigen Technology Inc., A Miltenyi Company: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Miltenyi Biotec B.V. & Co. KG: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.