Christian Brendel

Biochemical Correction of X-CGD by a Novel Chimeric Promoter Regulating High Levels of Transgene Expression in Myeloid Cells

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase catalytic subunit gp91(phox). A recent clinical trial for X-CGD using a spleen focus-forming virus (SFFV)-based γ-retroviral vector has demonstrated clear therapeutic benefits in several patients although complicated by enhancer-mediated mutagenesis and diminution of effectiveness over time due to silencing of the viral long terminal repeat (LTR). To improve safety and efficacy, we have designed a lentiviral vector that directs transgene expression primarily in myeloid cells. To this end, we created a synthetic chimeric promoter that contains binding sites for myeloid transcription factors CAAT box enhancer-binding family proteins (C/EBPs) and PU.1, which are highly expressed during granulocytic differentiation. As predicted, the chimeric promoter regulated higher reporter gene expression in myeloid than in nonmyeloid cells, and in human hematopoietic progenitors upon granulocytic differentiation. In a murine model of stem cell gene therapy for X-CGD, the chimeric vector resulted in high levels of gp91(phox) expression in committed myeloid cells and granulocytes, and restored normal NADPH-oxidase activity. These findings were recapitulated in human neutrophils derived from transduced X-CGD CD34(+) cells in vivo, and suggest that the chimeric promoter will have utility for gene therapy of myeloid lineage disorders such as CGD.

Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor.

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.

Mammary epithelial reconstitution with gene-modified stem cells assigns roles to Stat5 in luminal alveolar cell fate decisions, differentiation, involution, and mammary tumor formation.

The mammary gland represents a unique model system to study gene functions in adult stem cells. Mammary stem cells (MaSCs) can regenerate a functional epithelium on transplantation into cleared fat pads. We studied the consequences of distinct genetic modifications of MaSCs on their repopulation and differentiation ability. The reconstitution of ductal trees was used as a stem cell selection procedure and the nearly quantitative lentiviral infection efficiency of the primary mammary epithelial cells (MECs) rendered the enrichment of MaSCs before their transplantation unnecessary. The repopulation frequency of transduced MaSCs was nearly 100% in immunodeficient recipients and the resulting transgenic ducts homogeneously expressed the virally encoded fluorescent marker proteins. Transplantation of a mixture of MECs, expressing different fluorescent proteins, resulted in a distinct pattern of ductal outgrowths originating from a small number of individually transduced MaSCs. We used genetically modified MECs to define multiple functions of Stat5 during mammary gland development and differentiation. Stat5‐downregulation in MaSCs did not affect primary ductal outgrowth, but impaired side branching and the emergence of mature alveolar cells from luminal progenitors during pregnancy. Conversely, the expression of a constitutively active variant of Stat5 (cS5‐F) caused epithelial hyperproliferation, thickening of the ducts and precocious, functional alveoli formation in virgin mice. Expression of cS5‐F also prevented involution and caused the formation of estrogen and progesterone receptor positive (ER+PR+) adenocarcinomas. The tumors expressed activated Stat5 and Stat3 and contained a small fraction of CD44+ cells, possibly indicative of cancer stem cells. STEM CELLS 2010;28:928–938

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

Methylation‐induced gene silencing represents a major obstacle to efficient transgene expression in pluripotent cells and thereof derived tissues. As ubiquitous chromatin opening elements (UCOE) have been shown to prevent transgene silencing in cell lines and primary hematopoietic cells, we hypothesized a similar activity in pluripotent cells. This concept was investigated in the context of cytidine deaminase (CDD) gene transfer, an approach to render hematopoietic cells resistant to the chemotherapeutic agent Ara‐C. When murine induced pluripotent stem cells (iPSC)/embryonic stem cells (ESCs) were transduced with self‐inactivating lentiviral vectors using housekeeping (truncated elongation factor 1α; EFS) or viral (spleen focus‐forming virus; SFFV) promoters, incorporation of an heterogeneous nuclear ribonucleoproteins A2 B1/chromobox protein homolog 3 locus‐derived UCOE (A2UCOE) significantly increased transgene expression and Ara‐C resistance and effectively prevented silencing of the SFFV‐promoter. The EFS promoter showed relatively stable transgene expression in naïve iPSCs, but rapid transgene silencing was observed upon hematopoietic differentiation. When combined with the A2UCOE, however, the EFS promoter yielded stable transgene expression in 73% ± 6% of CD41+ hematopoietic progeny, markedly increased CDD expression levels, and significantly enhanced Ara‐C resistance in clonogenic cells. Bisulfite sequencing revealed protection from differentiation‐induced promoter CpG methylation to be associated with these effects. Similar transgene promoting activities of the A2UCOE were observed during murine neurogenic differentiation, in naïve human pluripotent cells, and during nondirected multilineage differentiation of these cells. Thus, our data provide strong evidence that UCOEs can efficiently prevent transgene silencing in iPS/ESCs and their differentiated progeny and thereby introduce a generalized concept to circumvent differentiation‐induced transgene silencing during the generation of advanced iPSC/ESC‐based gene and cell therapy products. STEM CELLS2013;31:488–499

Survival of the Fittest: Positive Selection of CD4+ T Cells Expressing a Membrane-Bound Fusion Inhibitor Following HIV-1 Infection

Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter

Protection against epigenetic silencing is a desirable feature of future gene therapy vectors, in particular for those applications in which transgene expression will not confer growth advantage to gene-transduced cells. The ubiquitous chromatin opening element (UCOE) consisting of the methylation-free CpG island encompassing the dual divergently transcribed promoters of the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) has been shown to shield constitutive active heterologous promoters from epigenetic modifications and chromosomal position effects. However, it is unclear if this element can be used to improve expression from tissue-specific enhancer/promoters, while maintaining tissue specificity in hematopoietic cells. Here, we evaluated the potential of the A2UCOE in combination with the myeloid-specific myeloid related protein 8 (MRP8) promoter to target transgene expression specifically to myeloid cells in vitro and in vivo from a self-inactivating lentiviral vector. The inclusion of the A2UCOE did not interfere with specific upregulation of MRP8 promoter activity during myeloid differentiation and mediated sustained and vector copy-dependent expression in myeloid cells. Notably, the A2UCOE did not protect the MRP8 promoter from methylation in the P19 embryonal carcinoma cell line, suggesting that this element maintains the inherent epigenetic state and transcriptional activity of cellular promoters in their native configuration. Thus, the A2UCOE could represent a useful protective genetic element in gene therapy vectors, ensuring physiological transcriptional regulation of tissue-specific promoters independent of the chromosomal integration site.

miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction

RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNA(miR)) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNA(miR)s resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNA(miR)s for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences.

Safer, Silencing-Resistant Lentiviral Vectors: Optimization of the Ubiquitous Chromatin-Opening Element through Elimination of Aberrant Splicing

ABSTRACT Gammaretroviral and lentiviral vectors have been used successfully in several clinical gene therapy trials, although powerful enhancer elements have caused insertional mutagenesis and clonal dysregulation. Self-inactivating vectors with internal heterologous regulatory elements have been developed as potentially safer and more effective alternatives. Lentiviral vectors containing a ubiquitous chromatin opening element from the human HNRPA2B1-CBX3 locus (A2UCOE), which allows position-independent, long-term transgene expression, are particularly promising. In a recently described assay, aberrantly spliced mRNA transcripts initiated in the vector A2UCOE sequence were found to lead to upregulation of growth hormone receptor gene (Ghr) expression in transduced murine Bcl-15 cells. Aberrant hybrid mRNA species formed between A2UCOE and a number of other cellular genes were also detected in transduced human PLB-985 myelomonocytic cells. Modification of the A2UCOE by mutation or deletion of recognized and potential cryptic splice donor sites was able to abrogate these splicing events and hybrid mRNA formation in Bcl-15 cells. This modification did not compromise A2UCOE regulatory activity in terms of resistance to CpG methylation and gene silencing in murine P19 embryonic carcinoma cells. These refined A2UCOE regulatory elements are likely to improve intrinsic biosafety and may be particularly useful for a number of clinical applications where robust gene expression is desirable.

A minimal ubiquitous chromatin opening element (UCOE) effectively prevents silencing of juxtaposed heterologous promoters by epigenetic remodeling in multipotent and pluripotent stem cells

Epigenetic silencing of transgene expression represents a major obstacle for the efficient genetic modification of multipotent and pluripotent stem cells. We and others have demonstrated that a 1.5 kb methylation-free CpG island from the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) effectively prevents transgene silencing and variegation in cell lines, multipotent and pluripotent stem cells, and their differentiated progeny. However, the bidirectional promoter activity of this element may disturb expression of neighboring genes. Furthermore, the epigenetic basis underlying the anti-silencing effect of the UCOE on juxtaposed promoters has been only partially explored. In this study we removed the HNRPA2B1 moiety from the A2UCOE and demonstrate efficient anti-silencing properties also for a minimal 0.7 kb element containing merely the CBX3 promoter. This DNA element largely prevents silencing of viral and tissue-specific promoters in multipotent and pluripotent stem cells. The protective activity of CBX3 was associated with reduced promoter CpG-methylation, decreased levels of repressive and increased levels of active histone marks. Moreover, the anti-silencing effect of CBX3 was locally restricted and when linked to tissue-specific promoters did not activate transcription in off target cells. Thus, CBX3 is a highly attractive element for sustained, tissue-specific and copy-number dependent transgene expression in vitro and in vivo.

Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype

Reducing expression of the fetal hemoglobin (HbF) repressor BCL11A leads to a simultaneous increase in γ-globin expression and reduction in β-globin expression. Thus, there is interest in targeting BCL11A as a treatment for β-hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia. Here, we found that using optimized shRNAs embedded within an miRNA (shRNAmiR) architecture to achieve ubiquitous knockdown of BCL11A profoundly impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs) despite a reduction in nonspecific cellular toxicities. BCL11A knockdown was associated with a substantial increase in S/G2-phase human HSCs after engraftment into immunodeficient (NSG) mice, a phenotype that is associated with HSC exhaustion. Lineage-specific, shRNAmiR-mediated suppression of BCL11A in erythroid cells led to stable long-term engraftment of gene-modified cells. Transduced primary normal or SCD human HSCs expressing the lineage-specific BCL11A shRNAmiR gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60%-70% γ-chain expression (vs. < 10% for negative control) and a corresponding increase in HbF. Transplantation of gene-modified murine HSCs from Berkeley sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key pathophysiological biomarkers of SCD. These data form the basis for a clinical trial application for treating sickle cell disease.