Self-inactivating Vector Research Articles

Gene-Corrected Fibroblast Therapy for Recessive Dystrophic Epidermolysis Bullosa using a Self-Inactivating COL7A1 Retroviral Vector

Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack type VII collagen and therefore have severely impaired dermal-epidermal stability causing recurrent skin and mucosal blistering. There is currently no specific approved treatment for RDEB. We present preclinical data showing that intradermal injections of genetically corrected patient-derived RDEB fibroblasts using a Good Manufacturing Practices grade self-inactivating COL7A1 retroviral vector reverse the disease phenotype in a xenograft model in nude mice. We obtained 50% transduction efficiency in primary human RDEB fibroblasts with an average low copy number (range= 1-2) of integrated provirus. Transduced fibroblasts showed strong type VII collagen re-expression, improved adhesion properties, normal proliferative capabilities, and viability invitro. We show that a single intradermal injection of 3× 10(6) genetically corrected RDEB fibroblasts beneath RDEB skin equivalents grafted onto mice allows type VII collagen deposition, anchoring fibril formation at the dermal-epidermal junction, and improved dermal-epidermal adherence 2 months after treatment, supporting functional correction invivo. Gene-corrected fibroblasts previously showed no tumorigenicity. These data show the efficacy and safety of gene-corrected fibroblast therapy using a self-inactivating vector that has now been good manufacturing grade-certified and pave the way for clinical translation to treat nonhealing wounds in RDEB patients.

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

Correction of Fanconi Anemia Mutation Using the Crispr/Cas9 System

IntroductionGene therapy has been developed for genetic diseases, either to restore normal function for loss-of-function mutations or to inhibit gain-of-function mutations. Gene addition using genetically engineered viral and plasmid vectors has successfully corrected cell pathophysiology resulting in the production of functional proteins. Therapeutic safety has been reinforced by the use of self-inactivating vectors; however, the potential risk of tumorigenesis raises concerns for insertional mutagenesis combined with acquired somatic mutations.Recent advances in gene editing using an RNA-guided endonuclease (RGEN), known as the CRISPR/Cas9 system, have opened a new frontier for the in situ correction of disease-associated mutations. Genomic DNA of cells harboring mutations can be excised and replaced with a DNA template for the functional gene sequence using homology-directed repair (HDR). The advantages of this repair include fewer off-target effects and a reduced risk of copy number changes compared with gene addition using vectors.Fanconi anemia (FA) is a syndrome of inherited bone marrow failure, characterized by the deficient regulation of DNA double-strand break repair. Clinical trials of gene therapy using viral vectors are still on-going with partial success; therefore, a new gene editing technique deserves attention. However, the feasibility of this approach in diseases with impaired HDR, such as FA, is unknown. Therefore, we used an RGEN to generate a cell line harboring a disease-causing point mutation in an FA-associated gene and elucidated the efficacy of restoring the mutation thereafter.MethodspSpCas9(BB) (PX330) was used to express humanized S. pyogenes Cas9 and single guide RNAs (sgRNAs) of interest. The sgRNAs were designed by searching for NGG protospacer adjacent motif (PAM) sequences near the point mutation target sites. The candidate sgRNAs were designed to be specific for the FANCC c.67delG:p.D23Ifs*23 mutation type (MT) or wild type (WT): gRNA#4, 5′-ATGGGATCAGGCTTCCACTT-3′ and gRNA#5, 5′-GAAGCTTTCTGTATGGGATC-3′ were specific for the WT sequence; whereas, gRNA M4, 5′-TATGGATCAGGCTTCCACTT-3′ and gRNA M5, 5′-AGAAGCTTTCTGTATGGATC-3′ were specific for the MT sequence. pCAG-EGxxFP, an EGFP-based reporter plasmid for the HDR that harbored the 500-bp target region of the WT or MT FANCC, was constructed for the gRNA selection. An HDR template construct was designed to incorporate a puromycin-resistant gene flanked by two loxP sites and two homologous arms containing the WT or MT sequence.HEK293T cells harboring the WT FANCC sequence were genetically edited by the above-mentioned plasmids.ResultsTo validate an efficient and specific sgRNA for DNA double-strand breaks, we cotransfected pCAG-EGxxFP-FANCC WT or MT and pSpCas9(BB)-FANCC-gRNA plasmids into HEK293T cells. EGFP fluorescence, whose intensity is correlated with the efficacy of HDR and thus the efficacy and specificity of sequence-specific DNA excision, was observed 48 h later, and we determined that gRNA#4 and gRNA M4 were specific for the WT and MT sequences, respectively.To generate cells harboring the MT FANCC sequence, HEK293T cells were cotransfected with pSpCas9(BB)-FANCC-gRNA#4 and the HDR template plasmid harboring the MT FANCC. A cell harboring biallelic MT FANCC was selected by adding puromycin and single-cell cloning. The transient expression of Cre recombinase in this clone successfully deleted the drug-selection cassette, and 293T-FANCC c.67delG cells were established. This cell showed the loss of FANCD2 monoubiquitination, a hallmark of a deficient FA core complex.Next, the 293T-FANCC c.67delG cells were cotransfected with pSpCas9(BB)-FANCC-gRNA M4 and the HDR template with the WT FANCC. This restoration of the mutated FANCC sequence resulted in a high frequency of at least monoallelic correction and the restoration of FANCD2 monoubiquitination.ConclusionsThe feasibility of genome editing was demonstrated in cells harboring an FA mutation, which can be a foothold for future therapy using precision gene restoration in patients with impaired HDR. DisclosuresNo relevant conflicts of interest to declare.

Kinetics of lentiviral vector transduction in human CD34+ cells

Unlike cell lines, human hematopoietic stem cells (HSCs) are less efficiently transduced with HIV-1 vectors, potentially limiting this approach. To investigate which step (internalization, reverse transcription, nuclear transport, and integration) limits lentiviral transduction, we evaluated the kinetics of lentiviral transduction in human CD34(+) cells. We transduced HeLa and CD34(+) cells with self-inactivating HIV-1 vector at low and tenfold higher multiplicity of infection (MOI) and evaluated vector amounts at various time points based on the rationale that if a given step was not limiting, tenfold greater vector amounts would be obtained at the tenfold higher MOI. We observed slower internalization (>60 min), a peak in reverse transcription at 24 hours, and completion of integration at 3 days in CD34(+) cells. In HeLa cells, there were approximately tenfold greater amounts at high MOI at all time points. When compared with HeLa cells, CD34(+) cells exhibited larger differences in vector amounts between high and low MOIs at 2-6 hours and a smaller difference at 12 hours to 10 days, revealing a limitation in human CD34(+) cell transduction around 12 hours, which corresponds to reverse transcription. In serial measurements of reverse transcription at 24 hours, vector amounts did not decrease once detected among CD34(+) cells. When using an HSC expansion medium, we observed less limitation for starting reverse transcription and more efficient transduction among CD34(+) cells in vitro and in xenografted mice. These data suggest that it is the initiation of reverse transcription that limits lentiviral transduction of human CD34(+) cells. Our findings provide an avenue for optimizing human CD34(+) cell transduction.

British Society for Gene and Cell Therapy Annual Conference Glasgow9–11th June 2015Conference Abstracts

British Society for Gene and Cell Therapy Annual Conference Glasgow9–11th June 2015Conference Abstracts

Direct interactions between human mesenchymal stem cells and ovarian cancer cells

To employ a co-culture system of human mesenchymal stem cells (MSC) and ovarian cancer cells to elucidate the cellular interactions among ovarian tumor microenvironment. MSC and ovarian cancer cells of SK-OV-3, NIH:OVCAR-3 and SCCOHT-1 were transfected with third-generation lentiviral self-inactivating (SIN) vector containing enhanced green fluorescent protein (eGFP) or mCherry gene. The interactions between MSC and ovarian cancer cells were examined by fluorescence microscopy with appropriate monochrome fluorescence filters and a FITC/TRIC dual band fluorescence filter. Flow cytometry of both cells revealed a differential expression of cell surface markers during co-culturing. Moreover, microarray was used for further analyses of gene variation during co-culturing of both MSC and ovarian cancer cells. MSCs were successfully isolated from umbilical cords. After 5-6 days co-culturing of lentivirus eGFP-transduced human MSC with mCherry-labeled ovarian cancer cells, there was a growth stimulation of cancer cells by MSC accompanied by an induction of CD90, partial CD73 and CD105 in cancer cells and CD326 in MSC. Microarray data also showed various significant gene variations during co-culturing. Human MSCs highly stimulate the growth and expression of CD90, partial CD73 and CD105 in ovarian cancer cells during co-culturing. And mutual cellular protein exchanges result in CD326 expression and gene alteration in MSC.

542. A Novel In Vitro/Vivo Selection/Suicide Strategy for Lentiviral Vectors

Genetic engineering of hematopoietic progenitor cells (HPC) by lentiviral-based vectors suffers from their low transduction rate with ~20% when aiming a single integration event. This amount of modified cells may not be sufficient for the correction of a number of diseases and therefore enrichment of transduced cells would be needed. We propose a novel in vitro/in vivo selection/suicide system based on the expression of the human reduced folate carrier 1 (hRFC1) and the human ribonucleoside-diphosphate reductase subunit M2 (RRM2). These genes are encoding for proteins which are responsible for drug resistances/sensitivities.The vector design is based on a bicistronic self-inactivating vector, meaning that the vector comprises the ubiquitin promoter (UBI) which drives the expression of EGFP in order to track transduced cells. The PGK promoter drives the expression of hRFC1 and the RRM2, which are connected through the cleavage peptide T2A.The hRFC1 is the main transport protein for the uptake of folates which are indispensable for purine synthesis and therefore for DNA synthesis. A greater expression level of hRFC1 should render cells more sensitive to the antifolate Methotrexate (suicide), which mainly enters the cells via the hRFC1. In contrast, it is possible to enrich for the transduced cells with Trimetrexate, which is an analog of Methotrexate. Its very lipophilic nature allows Trimetrexate to passively diffuse through cell membranes. As a result, transduced cells can escape the toxic effects from Trimetrexate due to their augmented transport of folic acid trough the overexpressed hRFC.In contrast, non-transduced cells are expected to show the opposite effects with higher resistance to Methotrexate but lower threshold killing to Trimetrexate.The second resistance gene RRM2 catalyzes the reduction of ribonucleotides into desoxyribonucleotides and its overexpression should lead to resistance to the drug hydroxyurea. When applying both selection drugs (Trimetrexate and Hydroxyurea) we expect an additive or even synergistic toxic effect on non-transduced cells while the transduced cells experience lower side effects.We are currently examining the concept proposed in vitro using various cell lines. Providing promising results, we would like to use the vector on CD34+ hematopoietic stem cells derived from umbilical cord blood. In addition, we would like to transplant immunocompromised NSG mice with a mixture of transduced and non-transduced CD34+ cells and then apply the Trimetrexate/Hydroxyurea in vivo to select and then Methotrexate in order to remove the transduced cell from the circulation.

RETRACTED: Novel and safer self-inactivating vectors for gene therapy of Wiskott-Aldrich Syndrome

The article entitled “Novel and safer self-inactivating vectors for gene therapy of Wiskott-Aldrich Syndrome”, by E. G. Coci1, T. Maetzig, D. Zychlinski, M. Rothe, J. D. Suerth, C. Klein and A.Schambach has been retracted, on the request of the authors.Kindly see Bentham Science Policy on Article retraction at the link given below:(https://www.benthamscience.com/journals/current-medical-imaging/author-guidelines/).It is a pre-requisite for authors to declare explicitly that their work is original and has not been published elsewhere. Authors are advised to properly cite the original source to avoid plagiarism and copyright violation. As such this article represents a severe abuse of the scientific publishing system. Bentham Science Publishers takes a very strong view on this matter and apologizes to the readers of the journal for any inconvenience this may cause.

Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors

The risk of insertional mutagenesis inherent to all integrating exogenous expression cassettes was the impetus for the development of various integration-defective lentiviral vector (IDLV) systems. These systems were successfully employed in a plethora of preclinical applications, underscoring their clinical potential. However, current production of IDLVs by transient plasmid transfection is not optimal for large-scale production of clinical grade vectors. Here, we describe the development of the first tetracycline-inducible stable IDLV packaging cell line comprising the D64E integrase mutant and the VSV-G envelope protein. A conditional self-inactivating (cSIN) vector and a novel polypurine tract (PPT)-deleted vector were incorporated into the newly developed stable packaging cell line by transduction and stable transfection, respectively. High-titer (~107 infectious units (IU)/ml) cSIN vectors were routinely generated. Furthermore, screening of single-cell clones stably transfected with PPT-deleted vector DNA resulted in the identification of highly efficient producer cell lines generating IDLV titers higher than 108 IU/mL, which upon concentration increased to 1010 IU/ml. IDLVs generated by stable producer lines efficiently transduce CNS tissues of rodents. Overall, the availability of high-titer IDLV lentivirus packaging cell line described here will significantly facilitate IDLV-based basic science research, as well as preclinical and clinical applications.

HEK293-based production platform for γ-retroviral (self-inactivating) vectors: application for safe and efficient transfer of COL7A1 cDNA.

The clinical application of self-inactivating (SIN) retroviral vectors requires an efficient vector production technology. To enable production of γ-retroviral SIN vectors from stable producer cells, new targetable HEK293-based producer clones were selected, providing amphotropic, GALV, or RD114 pseudotyping. Viral vector expression constructs can reliably be inserted at a predefined genomic locus via Flp-recombinase-mediated cassette exchange. Introduction of a clean-up step, mediated by Cre-recombinase, allows the removal of residual sequences that were required for targeting and selection, but were dispensable for the final producer clones and eliminated homology-driven recombination between the tagging and the therapeutic vector. The system was used to establish GALV and RD114 pseudotyping producer cells (HG- and HR820) for a clinically relevant long terminal repeat-driven therapeutic vector, designed for the transfer of a recombinant TCR that delivered titers in the range of 2×10(7) infectious particles (IP)/ml. Production capacity of the amphotropic producer cell (HA820) was challenged by a therapeutic SIN vector transferring the large COL7A1 cDNA. The final producer clone delivered a titer of 4×10(6) IP/ml and the vector containing supernatant was used directly to functionally restore primary fibroblasts and keratinocytes isolated from recessive dystrophic epidermolysis bullosa patients. Thus, the combinatorial approach (fc-technology) to generate producer cells for therapeutic γ-retroviral (SIN) vectors is feasible, is highly efficient, and allows their safe production and application in clinical trials.

A Modified γ-Retrovirus Vector for X-Linked Severe Combined Immunodeficiency

BackgroundIn previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus–based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancer-mediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1.MethodsWe enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc).ResultsAll patients received bone marrow–derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients.ConclusionsThis modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.)

Use of the piggyBac transposon to create stable packaging cell lines for the production of clinical-grade self-inactivating γ-retroviral vectors.

Efforts to improve the biosafety of γ-retroviral-mediated gene therapy have resulted in a shift toward the use of self-inactivating (SIN) γ-retroviral vectors. However, scale-up and manufacturing of such vectors requires significant optimization of transient transfection-based processes or development of novel platforms for the generation of stable producer cell clones. To that end, we describe the use of the piggybac transposon to generate stable producer cell clones for the production of SIN γ-retroviral vectors. The piggybac transposon is a universal tool allowing for the stable integration of SIN γ-retroviral constructs into murine (PG13) and human 293-based Phoenix (GALV and RD114, respectively) packaging cell lines without reverse transcription. Following transposition, a high-titer clone is selected for manufacture of a master cell bank and subsequent γ-retroviral vector supernatant production. Packaging cell clones created using the piggybac transposon have comparable titers to non-SIN vectors generated via conventional methods. We describe herein the use of the piggybac transposon for the production of stable packaging cell clones for the manufacture of clinical-grade SIN γ-retroviral vectors for ex vivo gene therapy clinical trials.

Toward a Safer Integration Profile of MLV-based Retroviral Vectors

Toward a Safer Integration Profile of MLV-based Retroviral Vectors

The U3 Region of Moloney Murine Leukemia Virus Contains Position-independent Cis-acting Sequences Involved in the Nuclear Export of Full-length Viral Transcripts

The distinguishing feature of self-inactivating (SIN) retroviral vectors is the deletion of the enhancer/promoter sequences in the U3 region of the 3' long terminal repeat. This design is used to overcome transcriptional interference and prevent downstream transcription from the 3' long terminal repeat. SIN vectors were derived from a number of different retroviruses. Studies of SIN vectors show that extensive U3 deletions in HIV-based vectors do not alter viral titers or the in vitro and in vivo properties of the vectors. However, deletion of the U3 sequences in γ- and α-retroviruses correlates with defects in 3' RNA processing and reduces viral titers by >10-fold. Here, we studied the steps in the retroviral life cycle that are affected by the deletion of sequences in the 3' U3 region of Moloney murine leukemia virus-derived retroviral vectors. The results show that the amounts of both full-length and internal RNA transcripts of U3-minus vectors are reduced in the nuclei of transfected cells, an effect that is probably due to a general defect in 3' RNA processing. Furthermore, full-length RNA transcripts were also defective in terms of nuclear export. This defect was complemented by transferring the U3 region to another position within the retroviral vector, indicating that the U3 region contains position-independent cis-acting sequences that are required for the transport of full-length viral transcripts. The results also suggest that the leader region of Moloney murine leukemia virus contains inhibitory/regulatory sequences, which prevent export and mediate nuclear retention of full-length viral RNA.

Gene therapy for primary immunodeficiencies: current status and future prospects.

Gene therapy using autologous haematopoietic stem cells offers a valuable treatment option for patients with primary immunodeficiencies who do not have access to an HLA-matched donor, although such treatments have not been without their problems. This review details gene therapy trials for X-linked and adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS) and chronic granulomatous disease (CGD). X-linked SCID was chosen for gene therapy because of previous 'natural' genetic correction through a reversion event in a single lymphoid precursor, demonstrating limited thymopoiesis and restricted T-lymphocyte receptor repertoire, showing selective advantage of progenitors possessing the wild-type gene. In early studies, patients were treated with long terminal repeats-intact gamma-retroviral vectors, without additional chemotherapy. Early results demonstrated gene-transduced cells, sustained thymopoiesis, and a diverse T-lymphocyte repertoire with normal function. Serious adverse effects were subsequently reported in 5 of 20 patients, with T-lymphocyte leukaemia developing, secondary to the viral vector integrating adjacent to a known oncogene. New trials using self-inactivating gamma-retroviral vectors are progressing. Trials for ADA-SCID using gamma-retroviral vectors have been successful, with no similar serious adverse effects reported; trials using lentiviral vectors are in progress. Patients with WAS and CGD treated with early gamma-retroviral vectors have developed similar lymphoproliferative adverse effects to those seen in X-SCID--current trials are using new-generation vectors. Targeted gene insertion using homologous recombination of corrected gene sequences by cellular DNA repair pathways following targeted DNA breakage will improve efficacy and safety of gene therapy. A number of new techniques are discussed.

Genome-wide analysis of alpharetroviral integration in human hematopoietic stem/progenitor cells.

Gene transfer vectors derived from gamma-retroviruses or lentiviruses are currently used for the gene therapy of genetic or acquired diseases. Retroviral vectors display a non-random integration pattern in the human genome, targeting either regulatory regions (gamma-retroviruses) or the transcribed portion of expressed genes (lentiviruses), and have the potential to deregulate gene expression at the transcriptional or post-transcriptional level. A recently developed alternative vector system derives from the avian sarcoma-leukosis alpha-retrovirus (ASLV) and shows favorable safety features compared to both gamma-retroviral and lentiviral vectors in preclinical models. We performed a high-throughput analysis of the integration pattern of self-inactivating (SIN) alpha-retroviral vectors in human CD34+ hematopoietic stem/progenitor cells (HSPCs) and compared it to previously reported gamma-retroviral and lentiviral vectors integration profiles obtained in the same experimental setting. Compared to gamma-retroviral and lentiviral vectors, the SIN-ASLV vector maintains a preference for open chromatin regions, but shows no bias for transcriptional regulatory elements or transcription units, as defined by genomic annotations and epigenetic markers (H3K4me1 and H3K4me3 histone modifications). Importantly, SIN-ASLV integrations do not cluster in hot spots and target potentially dangerous genomic loci, such as the EVI2A/B, RUNX1 and LMO2 proto-oncogenes at a virtually random frequency. These characteristics predict a safer profile for ASLV-derived vectors for clinical applications.

Lentiviral-based anti-HIV therapeutic vaccine: design, preclinical studies and phaseI/II clinical trial preliminary results

THERAVECTYS, a spin off the Pasteur Institute, develops a new generation of prophylactic and therapeutic vaccines using optimized lentiviral vectors. It’s most advanced product, a therapeutic anti-HIV vaccine treatment, has entered clinical Phase I/II end of 2012. This vaccination should allow seropositive patients to gain an immunological status identical to the so-called “Functional Cured” patients who develop an efficient immunological response capable of controlling the infection without therapy. Vaccine candidates are integrative and self-inactivated live-recombinant lentiviral vectors. They encode an HIV antigen, under the regulation of a patented promoter that is preferentially induced in APC (generating the specific immune response), and showing a basal level expression in all cells (allowing their elimination by the settled immune response).These vaccine candidates are classified as “Live recombinant vectored vaccines” (EMA, 2011). Preclinical studies demonstrated i)the generation of a strong, specific and very long lasting T-cell immune response (up to 2 years in murine animal models), ii) the restricted diffusion of the vaccine candidates after injection and iii)their fast disappearance within few weeks, correlated with an absence of macroscopic and microscopic toxicity. These data allowed the settlement of the anti-HIV therapeutic Phase I/II clinical trial that is held in France and Belgium and that has ended the enrollment of the 36 HIV-1 infected patients. THERAVECTYS’ anti-HIV vaccine treatment is assessed at three doses and safety, tolerability and immunogenicity compared to a placebo group. Furthermore, vaccine efficiency is be evaluated by the interruption of the HAART treatment in all patients, including placebo. Final results are expected by 2014 with intermediary analysis in April 2014.

Guest editorial: recent progress in gene therapy

After the occurrence of leukemia owing to insertional mutagenesis following hematopoietic stem cell gene therapy for severe immunodeficiencies using retroviral vectors, clinical trials of gene therapy remained stagnant for many years. However, clinical gene therapy has been revived because a number of successful clinical trials have been reported recently. For example, gene therapy of Leber’s congenital amaurosis, Parkinson’s disease, and hemophilia B, showed adeno-associated virus (AAV) vectors were safe and clinically effective gene delivery vehicles. AAV vectors can transduce non-dividing, terminally differentiated cells efficiently, and long-term transgene expression can be obtained after single gene transfer. Another superior gene delivery vector is the lentiviral vector, which was successfully used for hematopoietic stem cell gene therapy of X-linked adrenoleukodystrophy. Even in the case of classical hematopoietic stem cell gene therapy of severe immunodeficiencies, safety was much improved using selfinactivating retroviral vectors. Regarding cancer gene therapy, targeting technology is important to improve its efficacy. In this regard, there has been increasing focus on chimeric antigen receptor (CAR) technology when using engineered T cell therapy for hematological malignancies. CD19-targeted CAR-T gene therapy has had surprising clinical efficacy against chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In this ‘‘Progress in Hematology’’ issue, four excellent review articles [1–4] cover recent topics in the field of gene therapy. The first two articles deal with targeted cancer gene therapy, i.e., CAR technology [1] and mesenchymal stem cells (MSCs) with tumor-homing ability [2]. The third article describes gene therapy for hemophilia B [3], which is considered one of the best targets for AAV vectormediated gene therapy. The last article discusses hematopoietic stem cell gene therapy, the most representative and classical gene therapy for treatment of genetic diseases [4]. Davila et al. [1] provide a review on adoptive immunogene therapy using CAR-expressing T cells for hematological malignancies. Recent clinical trials of CD19-targeted CAR-T gene therapy revealed this therapeutic strategy is promising for the treatment of relapsed/refractory B cell malignancies, including CLL, ALL, and nonHodgkin lymphoma (NHL). Interestingly, robust and rapid anti-leukemia activity of CAR-T cells was observed in patients with B-ALL, while outcomes have been more modest in patients with CLL or NHL. Davila et al. also discuss the application of CAR technology for the treatment of multiple myeloma and acute myeloid leukemia. Another interesting targeting technology is the use of MSCs that accumulate at tumor sites. Uchibori et al. [2] reviewed the characteristics of MSCs and their potential as cellular vehicles for targeted cancer gene therapy. Since MSCs themselves may support tumor growth, genetical manipulation such as the addition of cytokine genes or suicide genes is required to provide them with anti-cancer activity. This review also discusses the molecular K. Ozawa (&) Division of Hematology, Department of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan e-mail: kozawa@ms2.jichi.ac.jp

Leptin gene therapy attenuates neuronal damages evoked by amyloid-β and rescues memory deficits in APP/PS1 mice

There is growing evidence that leptin is able to ameliorate Alzheimer's disease (AD)-like pathologies, including brain amyloid-β (Aβ) burden. In order to improve the therapeutic potential for AD, we generated a lentivirus vector expressing leptin protein in a self-inactivating HIV-1 vector (HIV-leptin), and delivered this by intra-cerebroventricular administration to APP/PS1 transgenic model of AD. Three months after intra-cerebroventricular administration of HIV-leptin, brain Aβ accumulation was reduced. By electron microscopy, we found that APP/PS1 mice exhibited deficits in synaptic density, which were partially rescued by HIV-leptin treatment. Synaptic deficits in APP/PS1 mice correlated with an enhancement of caspase-3 expression, and a reduction in synaptophysin levels in synaptosome preparations. Notably, HIV-leptin therapy reverted these dysfunctions. Moreover, leptin modulated neurite outgrowth in primary neuronal cultures, and rescued them from Aβ42-induced toxicity. All the above changes suggest that leptin may affect multiple aspects of the synaptic status, and correlate with behavioral improvements. Our data suggest that leptin gene delivery has a therapeutic potential for Aβ-targeted treatment of mouse model of AD.

A therapeutic approach towards chronic granulomatous disease

Chronic granulomatous disease (CGD) is a primary immunodeficiency (PID) characterized by the inability of phagocytes to produce reactive oxygen intermediates (ROIs) due to a defect in the NADPH oxidase complex. Recent studies have revealed that ROIs are involved in inflammatory signaling in phagocytes, illuminating the underlying mechanisms of hyper-inflammation in CGD. CGD patients frequently suffer from CGD-associated bowel inflammation, granuloma, and life-threatening infections. Based on the discovery of the regulatory function of ROIs in the immune response, therapeutic methods for excessive inflammation focusing on inflammatory cytokines are being developed for CGD. Although hematopoietic stem cell (HSC) transplantation (HSCT) is a curative therapy for CGD, successful transplants greatly depend on HSC source selection and the degree of matching of potential donors. Gene therapy trials for PID have been performed on over 120 patients with no HLA identical donor for HSCT, and have demonstrated clinical benefits. Genotoxicity in HSC gene therapy trials has expanded our knowledge on the mechanisms of vector-associated clonal expansion of gene-modified cells, which will advance gene therapy development using self-inactivating retrovirus and lentivirus vectors. We will discuss the complications of HSCT for CGD. We will then outline the status of gene therapy approaches in the treatment of CGD.