Molecular Evidence of Genome Editing in a Mouse Model of Immunodeficiency.

Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.

Regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis.

Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications that occur outside the endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of lysyl hydroxylase 3 (LH3, also known as PLOD3) into newly identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues.

Lentiviral labeling of mouse and human enteric nervous system stem cells for regenerative medicine studies.

BACKGROUND: Reliable methods of labeling human enteric nervous system (ENS) stem cells for use in novel cell replacement therapies for enteric neuropathies are lacking. Here, we explore the possibility of using lentiviral vectors expressing fluorescent reporter genes to transduce, label, and trace mouse and human ENS stem cells following transplantation into mouse gut. METHODS: Enteric nervous system precursors, including ENS stem cells, were isolated from enzymatically dissociated mouse and human gut tissues. Lentivirus containing eGFP or mCherry fluorescent reporter genes was added to gut cell cultures at a multiplicity of infection of 2-5. After fluorescence activated cell sorting for eGFP and subsequent analysis with markers of proliferation and cell phenotype, transduced mouse and human cells were transplanted into the gut of C57BL/6 and immune deficient Rag2-/gamma chain-/C5 mice, respectively and analyzed up to 60 days later. KEY RESULTS: Mouse and human transduced cells survived in vitro, maintained intense eGFP expression, proliferated as shown by BrdU incorporation, and formed characteristic neurospheres. When transplanted into mouse gut in vivo and analyzed up to 2 months later, transduced mouse and human cells survived, strongly expressed eGFP and integrated into endogenous ENS networks. CONCLUSIONS & INFERENCES: Lentiviral vectors expressing fluorescent reporter genes enable efficient, stable, long-term labeling of ENS stem cells when transplanted into in vivo mouse gut. This lentiviral approach not only addresses the need for a reliable fluorescent marker of human ENS stem cells for preclinical studies, but also raises the possibility of using lentiviruses for other applications, such as gene therapy.

Targeted expression of human folylpolyglutamate synthase for selective enhancement of methotrexate chemotherapy in osteosarcoma cells.

The antifolate methotrexate (MTX) is an important chemotherapeutic agent for treatment of osteosarcoma. This drug is converted intracellularly into polyglutamate derivates by the enzyme folylpolyglutamate synthase (FPGS). MTX polyglutamates show an enhanced and prolonged cytotoxicity in comparison to the monoglutamate. In the present study, we proved the hypothesis that transfer of the human fpgs gene into osteosarcoma cells may augment their MTX sensitivity. For this purpose, we employed the human osteocalcin (OC) promoter, which had shown marked osteosarcoma specificity in promoter studies using different luciferase assays in osteosarcoma and non-osteosarcoma cell lines. A recombinant lentiviral vector was generated with the OC promoter driving the expression of fpgs and the gene for enhanced green fluorescent protein (egfp), which was linked to fpgs by an internal ribosomal entry site (IRES). As the vector backbone contained only a self-inactivating viral LTR promoter, any interference of the OC promoter by unspecific promoter elements was excluded. We tested the expression of FPGS and enhanced green fluorescent protein (EGFP) after lentiviral transduction in various osteosarcoma cell lines (human MG-63 cells and TM 791 cells; rat osteosarcoma (ROS) 17/2.8 cells) and non-osteogenic tumor cell lines (293T human embryonic kidney cells, HeLa human cervix carcinoma cells). EGFP expression and MTX sensitivity were assessed in comparison with non-transduced controls. Whereas the OC promoter failed to enhance MTX sensitivity via FPGS expression in non-osteogenic tumor cell lines, the OC promoter mediated a markedly increased MTX cytotoxicity in all osteosarcoma cell lines after lentiviral transduction. The present chemotherapy-enhancing gene therapy system may have great potential to overcome in future MTX resistance in human osteosarcomas.

Correction of SCID-X1 using an enhancerless Vav promoter.

The efficacy of gene therapy for the treatment of inherited immunodeficiency has been highlighted in recent clinical trials, although in some cases complicated by insertional mutagenesis and silencing of vector genomes through methylation. To minimize these effects, we have evaluated the use of regulatory elements that confer reliability of gene expression, but also lack potent indiscriminate enhancer activity. The Vav1 proximal promoter is particularly attractive in this regard and may be useful in situations where high-level or complex regulation of gene expression is not necessary. X-linked severe combined immunodeficiency (SCID-X1) is a good candidate for such an approach, particularly as there may be additional disease-related intrinsic risks of leukemogenesis, and where safety is therefore a paramount concern. We have tested whether lentiviral vectors expressing the common cytokine receptor gamma chain under the control of the proximal Vav1 gene promoter are effective for correction of signaling defects and the disease phenotype. Despite low-level gene expression, we observed near-complete restoration of cytokine-mediated STAT5 phosphorylation in a model cell line. Furthermore, at low vector copy number, highly effective T- and B-lymphocyte reconstitution was achieved in vivo in a murine model of SCID-X1, in both primary and secondary graft recipients. This vector configuration deserves further evaluation and consideration for future clinical trials.

Efficient gene delivery to the adult and fetal CNS using pseudotyped non-integrating lentiviral vectors.

Non-integrating lentiviral vectors show considerable promise for gene therapy applications as they persist as long-term episomes in non-dividing cells and diminish risks of insertional mutagenesis. In this study, non-integrating lentiviral vectors were evaluated for their use in the adult and fetal central nervous system of rodents. Vectors differentially pseudotyped with vesicular stomatitis virus, rabies and baculoviral envelope proteins allowed targeting of varied cell populations. Efficient gene delivery to discrete areas of the brain and spinal cord was observed following stereotactic administration. Furthermore, after direct in utero administration (E14), sustained and strong expression was observed 4 months into adulthood. Quantification of transduction and viral copy number was comparable when using non-integrating lentivirus and conventional integrating vector. These data support the use of non-integrating lentiviral vectors as an effective alternative to their integrating counterparts in gene therapy applications, and highlight their potential for treatment of inherited and acquired neurological disorders.

Haematopoietic repopulating activity in human cord blood CD133+ quiescent cells.

We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133(+)G(1) cells. Here, we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures, the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (P<0.001) throughout the culture period. Furthermore, a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.

Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector.

An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/matrix attachment region (S/MAR) with a human liver-specific promoter (alpha1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. All relevant control pDNA constructs expressing luciferase are unable to sustain significant transgene expression beyond 1 week post-administration. We establish that this shutdown of expression is due to promoter methylation. In contrast, the S/MAR element appears to inhibit methylation of the AAT promoter thereby preventing transgene silencing. Although this vector appears to be maintained as an episome throughout, we have no evidence for its establishment as a replicating entity. We conclude that the combination of a mammalian, tissue-specific promoter with the S/MAR element is sufficient to drive long-term episomal pDNA expression of genes in vivo.

Lentiviral transduction of the murine lung provides efficient pseudotype and developmental stage-dependent cell-specific transgene expression.

Gene transfer for cystic fibrosis (CF) airway disease has been hampered by the lung's innate refractivity to pathogen infection. We hypothesized that early intervention with an integrating gene transfer vector capable of transducing the lung via the lumen may be a successful therapeutic approach. An HIV-based lentiviral vector pseudotyped with the baculovirus gp64 envelope was applied to the fetal, neonatal or adult airways. Fetal intra-amniotic administration resulted in transduction of approximately 14% of airway epithelial cells, including both ciliated and non-ciliated epithelia of the upper, mid and lower airways; there was negligible alveolar or nasal transduction. Following neonatal intra-nasal administration we observed significant transduction of the airway epithelium (approximately 11%), although mainly in the distal lung, and substantial alveolar transduction. This expression was still detectable at 1 year after application. In the adult, the majority of transduction was restricted to the alveoli. In contrast, vesicular stomatitis virus glycoprotein pseudotyped virus transduced only alveoli after adult and neonatal application and no transduction was observed after fetal administration. Repeat administration did not increase transduction levels of the conducting airway epithelia. These data demonstrate that application at early developmental stages in conjunction with an appropriately pseudotyped virus provides efficient, high-level transgene expression in the murine lung. This may provide a modality for treatment for lung disease in CF.

Cell-specific and efficient expression in mouse and human B cells by a novel hybrid immunoglobulin promoter in a lentiviral vector.

The expression of genes specifically in B cells is of great interest in both experimental immunology as well as in future clinical gene therapy. We have constructed a novel enhanced B cell-specific promoter (Igk-E) consisting of an immunoglobulin kappa (Igk) minimal promoter combined with an intronic enhancer sequence and a 3' enhancer sequence from Ig genes. The Igk-E promoter was cloned into a lentiviral vector and used to control expression of enhanced green fluorescent protein (eGFP). Transduction of murine B-cell lymphoma cell lines and activated primary splenic B cells, with IgK-E-eGFP lentivirus, resulted in expression of eGFP, as analysed by flow cytometry, whereas expression in non-B cells was absent. The specificity of the promoter was further examined by transducing Lin(-) bone marrow with Igk-E-eGFP lentivirus and reconstituting lethally irradiated mice. After 16 weeks flow cytometry of lymphoid tissues revealed eGFP expression by CD19+ cells, but not by CD3+, CD11b+, CD11c+ or Gr-1+ cells. CD19+ cells were comprised of both marginal zone B cells and recirculating follicular B cells. Activated human peripheral mononuclear cells were also transduced with Igk-E-eGFP lentivirus under conditions of selective B-cell activation. The Igk-E promoter was able to drive expression of eGFP only in CD19+ cells, while eGFP was expressed by both spleen focus-forming virus and cytomegalovirus constitutive promoters in CD19+ and CD3+ lymphocytes. These data demonstrate that in these conditions the Igk-E promoter is cell specific and controls efficient expression of a reporter protein in mouse and human B cells in the context of a lentiviral vector.

Ectopic retroviral expression of LMO2, but not IL2Rgamma, blocks human T-cell development from CD34+ cells: implications for leukemogenesis in gene therapy.

The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rgamma), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rgamma had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rgamma may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth.

The relationship between plasma concentration of valproic acid and its anticonvulsant and behavioural effects in the rat.

The relationship between the plasma concentration of valproic acid (VPA) and anticonvulsant or neurotoxic effects was studied in the rat. Anticonvulsant activity was assessed against; (1) maximal seizures induced either by electroshock or by intravenous injection of pentylenetetrazol; and (2) kindled amygdaloid epilepsy. Drug-induced neurotoxicity was determined by the rotarod test and by observation of behaviour. In the maximal seizure tests, tonic hindlimb extension was always abolished at plasma valproic acid concentrations of 225 microgram ml-1 and above. Tonic forelimb extension was not consistently blocked until the plasma drug concentration exceeded 530 microgram ml-1. In fully-kindled rats, plasma valproic acid concentrations of 300 microgram ml-1 and above markedly reduced the duration of amygdala afterdischarge activity and the intensity of behavioural seizures produced by amygdala stimulation. Analysis of the data from individual kindled rats revealed that there was a significant correlation between the estimated plasma concentration of valproic acid and the degree of seizure protection. Impairment of rotarod performance and marked ataxia occurred at plasma valproic acid concentrations above 510 microgram ml-1 and loss of righting reflex became evident at 970 microgram ml-1. From these results, it is concluded that the plasma concentration of valproic acid is closely correlated with the anticonvulsant and neurotoxic effects observed in individual rats after acute administration of sodium valproate.