In vivo CAR T-cell generation in nonhuman primates using lentiviral vectors displaying a multidomain fusion ligand.

ABSTRACT: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo. Here, we describe the incorporation of T-cell activation and costimulatory signals onto the surface of VivoVec particles (VVPs) in the form of a multidomain fusion protein and show enhanced in vivo transduction and improved CAR T-cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into nonhuman primates resulted in the robust generation of anti-CD20 CAR T cells and the complete depletion of B cells for >10 weeks. These data validate the VivoVec platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).

Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes.

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.

Optimizing lentiviral vector transduction of hematopoietic stem cells for gene therapy.

Autologous gene therapy using lentiviral vectors (LVs) holds promise for treating monogenetic blood diseases. However, clinical applications can be limited by suboptimal hematopoietic stem cell (HSC) transduction and insufficient quantities of available vector. We recently reported gene therapy for X-linked severe combined immunodeficiency using a protocol in which patient CD34+ cells were incubated with two successive transductions. Here we describe an improved protocol for LV delivery to CD34+ cells that simplifies product manipulation, reduces vector consumption, and achieves greater vector copy number (VCN) of repopulating HSCs in mouse xenotransplantation assays. Notable findings include the following: (1) the VCN of CD34+ cells measured shortly after transduction did not always correlate with the VCN of repopulating HSCs after xenotransplantation; (2) single-step transduction at higher CD34+ cell concentrations (2-4 × 106/ml) conserved LV without compromising HSC VCN; (3) poloxamer F108 (LentiBOOST) increased HSC VCN by two- to threefold (average from three donors); (4) although LentiBOOST + prostaglandin E2 combination further increased VCN in vitro, the VCN observed in vivo were similar to LentiBOOST alone; (5) cyclosporine H increased the HSC VCN to a similar or greater extent with LentiBOOST in vivo. Our findings delineate an improved protocol to increase the VCN of HSCs after CD34+ cell transduction with clinically relevant LVs.

Coupling endonucleases with DNA end-processing enzymes to drive gene disruption.

Targeted DNA double-strand breaks introduced by rare-cleaving designer endonucleases can be harnessed for gene disruption applications by engaging mutagenic nonhomologous end-joining DNA repair pathways. However, endonuclease-mediated DNA breaks are often subject to precise repair, which limits the efficiency of targeted genome editing. To address this issue, we coupled designer endonucleases to DNA end-processing enzymes to drive mutagenic break resolution, achieving up to 25-fold enhancements in gene disruption rates.

Tracking genome engineering outcome at individual DNA breakpoints.

Site-specific genome engineering technologies are increasingly important tools in the postgenomic era, where biotechnological objectives often require organisms with precisely modified genomes. Rare-cutting endonucleases, through their capacity to create a targeted DNA strand break, are one of the most promising of these technologies. However, realizing the full potential of nuclease-induced genome engineering requires a detailed understanding of the variables that influence resolution of nuclease-induced DNA breaks. Here we present a genome engineering reporter system, designated 'traffic light', that supports rapid flow-cytometric analysis of repair pathway choice at individual DNA breaks, quantitative tracking of nuclease expression and donor template delivery, and high-throughput screens for factors that bias the engineering outcome. We applied the traffic light system to evaluate the efficiency and outcome of nuclease-induced genome engineering in human cell lines and identified strategies to facilitate isolation of cells in which a desired engineering outcome has occurred.

Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome.

The immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) leads to life-threatening hematopoietic cell dysfunction. We used WAS protein (WASp)-deficient mice to analyze the in vivo efficacy of lentiviral (LV) vectors using either a viral-derived promoter, MND, or the human proximal WAS promoter (WS1.6) for human WASp expression. Transplantation of stem cells transduced with MND-huWASp LV resulted in sustained, endogenous levels of WASp in all hematopoietic lineages, progressive selection for WASp+ T, natural killer T and B cells, rescue of T-cell proliferation and cytokine production, and substantial restoration of marginal zone (MZ) B cells. In contrast, WS1.6-huWASp LV recipients exhibited subendogenous WASp expression in all cell types with only partial selection of WASp+ T cells and limited correction in MZ B-cell numbers. In parallel, WS1.6-huWASp LV recipients exhibited an altered B-cell compartment, including higher numbers of λ-light-chain+ naive B cells, development of self-reactive CD11c+FAS+ B cells, and evidence for spontaneous germinal center (GC) responses. These observations correlated with B-cell hyperactivity and increased titers of immunoglobulin (Ig)G2c autoantibodies, suggesting that partial gene correction may predispose toward autoimmunity. Our findings identify the advantages and disadvantages associated with each vector and suggest further clinical development of the MND-huWASp LV for a future clinical trial for WAS.

Novel reporter systems for facile evaluation of I-SceI-mediated genome editing.

Two major limitations to achieve efficient homing endonuclease-stimulated gene correction using retroviral vectors are low frequency of gene targeting and random integration of the targeting vectors. To overcome these issues, we developed a reporter system for quick and facile testing of novel strategies to promote the selection of cells that undergo targeted gene repair and to minimize the persistence of random integrations and non-homologous end-joining events. In this system, the gene target has an I-SceI site upstream of an EGFP reporter; and the repair template includes a non-functional EGFP gene, the positive selection transgene MGMTP140K tagged with mCherry, and the inducible Caspase-9 suicide gene. Using this dual fluorescent reporter system it is possible to detect properly targeted integration. Furthermore, this reporter system provides an efficient approach to enrich for gene correction events and to deplete events produced by random integration. We have also developed a second reporter system containing MGMTP140K in the integrated target locus, which allows for selection of primary cells with the integrated gene target after transplantation. This system is particularly useful for testing repair strategies in primary hematopoietic stem cells. Thus, our reporter systems should allow for more efficient gene correction with less unwanted off target effects.

Developmentally regulated expression of MEF2C limits the response to BCR engagement in transitional B cells.

Transitional and naïve mature peripheral B cells respond very differently to B-cell receptor (BCR) cross-linking. While transitional B cells undergo apoptosis upon BCR engagement, mature B cells survive and proliferate. This differential response correlates with the capacity of mature, but not transitional B cells to transcribe genes that promote cell survival and proliferation, including those encoding c-Myc and the Bcl-2 family members Bcl-xL and A1. We recently demonstrated that transitional B cells fail to assemble transcriptional machinery at the promoter region of these target genes despite equivalent cytoplasmic signaling and nuclear translocation of key transcription factors including NF-κB and nuclear factor of activated T cells (NFAT). The transcription factor myocyte enhancer factor-2C (MEF2C) is regulated by both calcineurin and mitogen-activated protein kinase signaling pathways, and is essential for proliferation and survival downstream of BCR engagement in mature B cells. In this work, we demonstrate that transitional B cells have intrinsically low levels of MEF2C protein and DNA-binding activity, and that this developmental difference in MEF2C expression is functionally significant. Forced expression of MEF2C in transitional B cells promoted cell survival, proliferation, and upregulation of pro-survival genes. Thus, low MEF2C expression limits transitional B-cell responsiveness to BCR engagement before these cells reach maturity.

Targeted entry via somatostatin receptors using a novel modified retrovirus glycoprotein that delivers genes at levels comparable to those of wild-type viral glycoproteins.

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.

Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors.

A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications.

Preclinical proof of concept for VivoVec, a lentiviral-based platform for in vivo CAR T-cell engineering.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation. METHODS: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice. RESULTS: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy. CONCLUSIONS: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.

B cell-specific lentiviral gene therapy leads to sustained B-cell functional recovery in a murine model of X-linked agammaglobulinemia.

The immunodeficiency disorder, X-linked agammaglobulinemia (XLA), results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA, we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Emu) and Igbeta (B29) minimal promoter to drive B lineage-specific human Btk expression in Btk/Tec(-/-) mice, a strain that reproduces the features of human XLA. After transplantation of EmuB29-Btk-LV-transduced stem cells, treated mice showed significant, albeit incomplete, rescue of mature B cells in the bone marrow, peripheral blood, spleen, and peritoneal cavity, and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression, viral integration, and partial functional responses, consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage-targeted LV gene therapy in human XLA.

Development of B-lineage predominant lentiviral vectors for use in genetic therapies for B cell disorders.

Sustained, targeted, high-level transgene expression in primary B lymphocytes may be useful for gene therapy in B cell disorders. We developed several candidate B-lineage predominant self-inactivating lentiviral vectors (LV) containing alternative enhancer/promoter elements including: the immunoglobulin ß (Igß) (B29) promoter combined with the immunoglobulin µ enhancer (EµB29); and the endogenous BTK promoter with or without Eµ (EµBtkp or Btkp). LV-driven enhanced green fluorescent protein (eGFP) reporter expression was evaluated in cell lines and primary cells derived from human or murine hematopoietic stem cells (HSC). In murine primary cells, EµB29 and EµBtkp LV-mediated high-level expression in immature and mature B cells compared with all other lineages. Expression increased with B cell maturation and was maintained in peripheral subsets. Expression in T and myeloid cells was much lower in percentage and intensity. Similarly, both EµB29 and EµBtkp LV exhibited high-level activity in human primary B cells. In contrast to EµB29, Btkp and EµBtkp LV also exhibited modest activity in myeloid cells, consistent with the expression profile of endogenous Bruton's tyrosine kinase (Btk). Notably, EµB29 and EµBtkp activity was superior in all expression models to an alternative, B-lineage targeted vector containing the EµS.CD19 enhancer/promoter. In summary, EµB29 and EµBtkp LV comprise efficient delivery platforms for gene expression in B-lineage cells.

Effective, safe, and sustained correction of murine XLA using a UCOE-BTK promoter-based lentiviral vector.

X-linked agammaglobulinemia (XLA) is an immune disorder caused by mutations in Bruton's tyrosine kinase (BTK). BTK is expressed in B and myeloid cells, and its deficiency results in a lack of mature B cells and protective antibodies. We previously reported a lentivirus (LV) BTK replacement therapy that restored B cell development and function in Btk and Tec double knockout mice (a phenocopy of human XLA). In this study, with the goal of optimizing both the level and lineage specificity of BTK expression, we generated LV incorporating the proximal human BTK promoter. Hematopoietic stem cells from Btk -/- Tec -/- mice transduced with this vector rescued lineage-specific expression and restored B cell function in Btk -/- Tec -/- recipients. Next, we tested addition of candidate enhancers and/or ubiquitous chromatin opening elements (UCOEs), as well as codon optimization to improve BTK expression. An Eµ enhancer improved B cell rescue, but increased immunoglobulin G (IgG) autoantibodies. Addition of the UCOE avoided autoantibody generation while improving B cell development and function and reducing vector silencing. An optimized vector containing a truncated UCOE upstream of the BTK promoter and codon-optimized BTK cDNA resulted in stable, lineage-regulated BTK expression that mirrored endogenous BTK, making it a strong candidate for XLA therapy.

Genome editing of HBG1 and HBG2 to induce fetal hemoglobin.

Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress γ-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and ß-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rγ-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.

Successful targeting and disruption of an integrated reporter lentivirus using the engineered homing endonuclease Y2 I-AniI.

Current antiviral therapy does not cure HIV-infected individuals because the virus establishes lifelong latent infection within long-lived memory T cells as integrated HIV proviral DNA. Here, we report a new therapeutic approach that aims to cure cells of latent HIV infection by rendering latent virus incapable of replication and pathogenesis via targeted cellular mutagenesis of essential viral genes. This is achieved by using a homing endonuclease to introduce DNA double-stranded breaks (dsb) within the integrated proviral DNA, which is followed by triggering of the cellular DNA damage response and error-prone repair. To evaluate this concept, we developed an in vitro culture model of viral latency, consisting of an integrated lentiviral vector with an easily evaluated reporter system to detect targeted mutagenesis events. Using this system, we demonstrate that homing endonucleases can efficiently and selectively target an integrated reporter lentivirus within the cellular genome, leading to mutation in the proviral DNA and loss of reporter gene expression. This new technology offers the possibility of selectively disabling integrated HIV provirus within latently infected cells.

The block to membrane fusion differs with the site of ligand insertion in modified retroviral envelope proteins.

Efforts to achieve cell type-specific transduction of retroviral vectors for gene therapy have centred on modification of the envelope protein (Env). Typically, addition of a ligand to Env gives binding to the new or target receptor, but little or no infection, and affects the subunit association of the modified Env. We previously discovered two point mutations that increase targeted infection by over 1000-fold when added to an Env modified by N-terminal insertion of the receptor-binding domain from amphotropic murine leukemia virus Env. Here, we asked whether these mutations would similarly increase transduction by Env modified with a clinically relevant ligand, human interleukin-13 (IL-13L). Addition of the point mutations stabilized the weak subunit association observed in some IL-13L-modified Env proteins, but infection via the target IL-13 receptor still did not occur. Fluorescence-based cell-cell fusion assays and studies with a membrane-curving agent revealed that defects in membrane fusion differed with the site of ligand insertion. When IL-13 was inserted into the N terminus of Env, membrane fusion was blocked prior to membrane-lipid mixing, regardless of whether flanking flexible linkers were added. Unexpectedly, insertion of IL-13 in the proline-rich region showed evidence of initiation of fusion and fusion-peptide exposure, but fusion was blocked at a subsequent step prior to fusion-pore formation. Thus, the site of ligand insertion influenced initiation of membrane fusion and its progression. These observations suggest that a novel site for ligand insertion must be identified before clinically useful targeted transduction will be achieved.

An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation.

Pathogenic activation of the LMO2 proto-oncogene by an oncoretroviral vector insertion in a clinical trial for X-linked severe combined immunodeficiency (X-SCID) has prompted safety concerns. We used an adeno-associated virus vector to achieve targeted insertion of a gamma-retroviral long terminal repeat (LTR) driving a GFP expression cassette with flanking loxP sites in a human T-cell line at the precise location of vector integration in one of the patients with X-SCID. The LTR-GFP cassette was inserted into the first intron of the LMO2 gene, resulting in strong activation of LMO2. Cre-mediated cassette exchange was used to replace the original LTR-GFP cassette with one flanked by insulator elements leading to a several fold reduction in LMO2 expression. The LTR-GFP cassette was also replaced with a globin gene regulatory cassette that failed to activate the LMO2 gene in lymphoid cells. A gamma-retroviral vector with 2 intact LTRs resulted in activation of the LMO2 gene when inserted into the first intron, but a self-inactivating lentiviral vector with an internal cellular promoter and flanking insulator elements did not activate the LMO2 gene. Thus, this system is useful for comparing the safety profiles of vector cassettes with various regulatory elements for their potential for proto-oncogene activation.

A chromatin insulator blocks interactions between globin regulatory elements and cellular promoters in erythroid cells.

Retroviral vectors have been developed for gene therapy of blood disorders because they achieve long-term expression of the transgene. However, interactions between the regulatory elements contained in such vectors and cellular genes may result in pathogenic proto-oncogene activation. We designed a cassette consisting of a splice acceptor followed by the coding sequences for Green Fluorescent Protein (GFP) and a polyadenylation site which was inserted in a reverse orientation in self-inactivating lentiviral vectors. Retroviral vectors integrate preferentially in genes and in one-half of the integrations, the expression cassette will be in a tandem orientation with respect to the gene's promoter and potentially expressed when the trapped promoter is intrinsically active or activated by regulatory elements within the vector. Approximately 10% of the integrations of the control vector with only the GFP cassette resulted in expression of the GFP marker. The trapping cassette in vectors containing globin regulatory elements was expressed more frequently in erythroleukemia (K562) cells than the control cassette only vector but there was no increase in promoter trapping efficiency in HeLa cells. In contrast, we found that addition of a gammaretroviral long terminal repeat increased the frequency of GFP expression both in K562 and HeLa cells. Promoter activation in K562 cells by vectors containing globin regulatory elements was significantly reduced by addition of flanking insulator elements.