Gene Transduction Method Research Articles

Alternative Anesthesia of Neonatal Mice for Global rAAV Delivery in the Brain With Non-detectable Behavioral Interference in Adults.

Viral-transduced gene expression is the current standard for cell-type-specific labeling and cell tacking in experimental neuroscience. To achieve widespread gene expression, a viral delivery method to neonatal rodents was introduced more than two decades ago. Most of those neonatal viral vector injection-based gene transduction methods in mice used deep hypothermia for anesthesia, which was reported to be associated with behavioral impairments. To explore other options for neonatal viral applications, we applied a combination of Medetomidine, Midazolam, and Fentanyl (MMF), each of which can be antagonized by a specific antagonist. Later in their adulthood, we found that adult mice, that received the MMF-induced anesthesia, combined with virus-injected into the brain at postnatal day 2, showed similar performance in all behavioral tasks tested, including tasks for motor coordination, anxiety-related tasks, and spatial memory when compared to adult naïve littermates. This demonstrates that MMF anesthesia could be safely applied to mice for neonatal viral transduction at P2.

Optimization of piggybac transposon system for chimeric antigen receptor-modified T cells

Objective To establish a non-viral gene transduction method of T cells, providing a gene modification strategy for CAR-T cell immunotherapy. Methods CD19CAR cassette (1 470 bp) was inserted into XbaⅠ and EcoRⅠ sits of PiggyBac transposon vector PB513B-1, recombinant vector was named as pb19CAR. Dual plasmids (pb19CAR and transposase) were transducted into primary T cells using electroporation (1×20 ms 830 V/840 V/850 V) and nucleofection (U014/T023). The transduction efficiency and cell viability were compared and optimized. T cells were activated by plates coated with human CD19 (5 μg/ml) protein or CD3/CD28 mAb (5 μg/ml), and then amplified by interleukin (IL)-2 (500 U/ml) or mixed cytokines IL-7 (10 ng/ml)/IL-15 (10 ng/ml)/IL-21 (20 ng/ml) CD19CAR expression and phenotype of T cells were compared after activation and amplification. Results PiggyBac transposon vector (pb19CAR) was confirmed by XbaⅠ and EcoRⅠ enzyme digestion and gene sequencing analysis. Based on the flow cytometry and cell vitality detection, the transduction efficiency and cell viability of primary T cells by electroporation were higher than that of nucleofaction (51.0±2.3)% vs. (36.5±1.8)%, (75.0±3.8)% vs. (60.0±3.0)% (P=0.000). After amplification of 12 days post-electroporation, CD19CAR expression on T cells in CD19 protein activation group was significantly higher than that in CD3/CD28 mAb activation group (70.0±3.5)% vs. (54.0±2.7)% (P=0.000). The cell phenotypic analysis showed that the proportion of Tscm/Tscm-like in mixed cytokine group was a higher than that in IL-2 group (25.00±1.25)% vs. (12.20±0.60)%, and so is the Tcm cells (10.20±0.51)% vs. (3.00±0.15)% (P=0.000). Conclusion The method of pb19CAR/transposase electroporation reveals higher transduction efficiency and cell viability. The activation by target antigen CD19 protein contributes to the preferential amplification of CAR-T cells. The mixed cytokine group (IL-7/IL-15/IL-21) supports the growth of CAR-T cells with central memory and stem memory phenotypes. Key words: PiggyBac transposon; Chimeric antigen receptor; Electroporation

A novel method of gene transduction to the murine endometrium using in vivo electroporation.

To investigate the molecular pathways involved in successful embryo implantation in mammals, we developed a novel method for gene transduction into the murine endometrium using in vivo electroporation. Plasmid DNA with an enhanced green fluorescence protein (EGFP) gene was injected into the uterine cavity of non-pregnant female mice, and electrical pulses were subsequently applied to the uterine horn using plate electrodes. EGFP expression was found only in the uterine luminal epithelium (LE), but not in the stroma. EGFP fluorescence in the LE was limited to the site where the positive side of the electrodes was placed during electric stimulation. These results demonstrated that our novel method enabled us to transduce a gene into a desired location of the murine uterus.

Gene-manipulated Adipocytes for the Treatment of Various Intractable Diseases.

Although protein replacement is an effective treatment for serum protein deficiencies such as diabetes and hemophilia, recombinant protein products are not available for all rare inherited diseases due to the instability of the recombinant proteins and/or to cost. Gene therapy is the most attractive option for treating patients with such rare diseases. To develop an effective ex vivo gene therapy-based protein replacement treatment requires recipient cells that differ from those used in standard gene therapy, which is performed to correct the function of the recipient cells. Adipose tissue is an expected source of proliferative cells for cell-based therapies, including regenerative medicine and gene transfer applications. Based on recent advances in cell biology and extensive clinical experience in transplantation therapy for adipose tissue, we focused on the mature adipocyte fraction, which is the floating fraction after collagenase digestion and centrifugation of adipose tissue. Proliferative adipocytes were propagated from the floating fraction by the ceiling culture technique. These cells are designated as ceiling culture-derived proliferative adipocytes (ccdPAs). We first focused on lecithin:cholesterol acyltransferase (LCAT) deficiency, an inherited metabolic disorder caused by lcat gene mutation, and ccdPAs as a therapeutic gene vehicle for LCAT replacement therapy. In our recent in vitro and animal model studies, we developed an adipose cell manipulation procedure using advanced gene transduction methods and transplantation scaffolds. We herein introduce the progress made in novel adipose tissue-based therapeutic strategies for the treatment of protein deficiencies and describe their future applications for other intractable diseases.

Newly Developed Measles Viral Vector Can Efficiently Transduce Multiple Genes into naïve T Cells

Because blood cells can be obtained with relatively easy and safe procedure, they have been routinely used for transfusion and transplantation purposes. And they are now considered as attractive cell sources for developing new gene therapies including cancer therapy using various immune cells, and regenerative therapy using hematopoietic stem cells or induced pluripotent stem cells (iPS) cells. For example, chimeric antigen receptor modified autologous T cells have been considered as effective therapy for various cancers. And iPS cells have been easily established from peripheral T cells for the purpose of treating various diseases. However, in spite of these possibilities, the development of the safer and more efficient genetic modification methods of hematopoietic cells is imminent. In this study, we developed the novel measles viral (MV) vector which enables us to transduce multiple genes into immune cells. The wild type measles virus is one of the aerosol-transmitted viruses and has strong infectious capacity to immune cells, and epithelial cells via signaling lymphocyte activation molecule (SLAM) or nectin-4. First, we modified the wild type measles virus genome to non-transmissible and non-lytic, and equipped with the ability of transducing multiple genes, at most six genes, into target cells. Briefly, the intrinsically non-segmented wild type virus genome was divided into two segments and point mutations were introduced into the virus genes encoding hemagglutinin and the matrix protein. Moreover, as the fusion protein gene was removed from the virus genome, the virus could not replicate in neighbor cells. We examined the gene transduction efficiency of the gene modified measles virus (H8-Fd-MV vector) into hematopoietic cells. We first constructed the H8-Fd-MV vector with GFP gene and transduced into hematopoietic progenitor cells and immune cells from human cord blood and peripheral blood. We observed that almost all of HPCs from cord blood (99.7% in floating cells expressed CD34), T cells (99.9% in CD3+ cells), and B cells (98.2% in CD19+ cells) from peripheral blood expressed GFP at two days after the transduction. Especially, to express GFP gene in human peripheral T cells, it was not necessary to pre-stimulate them with CD3/CD28 beads (99.6% in stimulating T cells (72.9% in SLAM+ cells) v.s. 82.6 % in non-stimulating cells (37.4% in SLAM+ cells)). T cells from cord blood showed almost all naïve phenotype (CD4+ cells: 93.2±1.8% in CD45RA+CCR7+ cells, 1.7±1.1% in CD45RA+CCR7- cells; CD8+ cells: 41.6±5.7% in CD45RA+CCR7+ cells, and 41.9±11.0% in CD8+CD45RA+CCR7- cells) and T cells transduced by MV vector expressed GFP more (CD4+ cells: 80.3±13.7%, and CD8+ cells: 82.5±8.5%) than those transduced by Sendai viral vector (CD4+ cells: 15.5±0.7%, and CD8+ cells: 17.4±5.4%). These data suggested that H8-Fd-MV vector could transduce GFP gene efficiently into various T cell lineages including naïve T cells, which had been difficult to be transduced with classical gene transduction methods. We next generated H8-Fd-MV vector for expressing 6 genes (OCT4, SOX2, KLF4, L-MYC, PIN1, and GFP) and transduced into stimulated T cells. After 3 days, GFP+ T cells expressed all of these 6 genes. We also detected that more than 50% of the stimulated T cells with IL-2 expressed GFP at 14 days after the transduction. After 27 days from transfection, embryonic stem cell (ES cell)-like colonies were picked up and analyzed the character. These cells showed ES cell morphology over 20 times passages and expressed pluripotent marker (NANOG, OCT4, Tra-1-60, Tra-1-81). We also found T cell receptor rearrangements in these cells. Embryoid bodies from these cells expressed three germ line markers in vitro. We next examined the hematopoietic differentiation of these cells using coculture system with murine embryonic aorta-gonad-mesonephros region-derived stromal cell line (AGM-3 cells). The co-cultured cells harvested at day 12 expressed CD34 and CD45. These data suggested that we established iPS cells from terminal differentiated T cells using H8-Fd-MV vector for expressing reprogramming factor.These results indicated that multiple genes were expressed efficiently in immune cells using H8-Fd-MV vector. Highly efficient transduction ability of MV vector for T cells would enable us to develop new gene therapy targeting cancer using gene modified T cells as well as organ regeneration using iPS cells. DisclosuresNo relevant conflicts of interest to declare.

Tumor vessel-injuring ability improves antitumor effect of cytotoxic T lymphocytes in adoptive immunotherapy

Angiogenesis is required for normal physiologic processes, but it is also involved in tumor growth, progression and metastasis. Here, we report the development of an immune-based antiangiogenic strategy based on the generation of T lymphocytes that possess killing specificity for cells expressing vascular endothelial growth factor receptor 2 (VEGFR2). To target VEGFR2-expressing cells, we engineered cytotoxic T lymphocyte (CTL) expressing chimeric T-cell receptors (cTCR–CTL) comprised of a single-chain variable fragment (scFv) against VEGFR2 linked to an intracellular signaling sequence derived from the CD3ζ chain of the TCR and CD28 by retroviral gene transduction methods. The cTCR–CTL exhibited efficient killing specificity against VEGFR2 and a tumor-targeting function in vitro and in vivo. Reflecting such abilities, we confirmed that the cTCR–CTL strongly inhibited the growth of a variety of syngeneic tumors after adoptive transfer into tumor-bearing mice without consequent damage to normal tissue. In addition, CTL expressing both cTCR and tumor-specific TCR induced complete tumor regression due to enhanced tumor infiltration by the CTL and long-term antigen-specific function. These findings provide evidence that the tumor vessel-injuring ability improved the antitumor effect of CTLs in adoptive immunotherapy for a broad range of cancers by inducing immune-mediated destruction of the tumor neovasculature.

Emerging Methods for Preparing iPS Cells

In 1998, human embryonic stem cells were first generated and were expected to contribute greatly to regenerative medicine. However, when medical treatments were performed using human embryonic stem cells, there were problems, such as transplant rejection, as well as bioethical issues. Induced pluripotent stem cells were generated from mouse and human fibroblasts in 2006 and 2007 by introducing four transcription factors (Oct3/4, Sox2, c-Myc and Klf4). This process was defined as direct reprogramming, and induced pluripotent stem cells were better tolerated. Although induced pluripotent stem cells have contributed greatly to biomedical research and regenerative medicine, high tumorigenic potential is still a critical problem due to the introduction of the oncogene c-Myc and reprogramming with a virus vector. To address this, we reprogrammed somatic cells by transfection with microribonucleic acids to avoid using virus vectors for genomic integration into the host genome. We found that it was possible to reprogram mouse and human cells to pluripotency by direct transfection of three mature microribonucleic acids (mir-200c, -302s and -369s) with increased expression levels in embryonic stem cells and induced pluripotent stem cells. The microribonucleic acid-induced pluripotent stem cells have a reduced risk of mutations and tumorigenesis. Our laboratory also introduced four transcription factors (Oct3/4, Sox2, c-Myc and Klf4) into cancer cells, generating induced pluripotent cancer cells that exhibited strikingly less malignant features, suggesting the possibility of a novel type of cancer therapy. However, the gene transduction method is not yet safe for clinical applications, due to a genomic integration that may cause tumor formation. We are currently investigating the reprogramming method using microribonucleic acids in cancer cells to develop a very safe, highly efficient and highly complete reprogramming for clinical applications.

Optimization of Adenovirus Vectors for Transduction in Embryonic Stem Cells and Induced Pluripotent Stem Cells

Because embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can differentiate into various types of cells in vitro, they are considered as a valuable model to understand the processes involved in the differentiation into functional cells as well as an unlimited source of cells for therapeutic applications. Efficient gene transduction method is one of the powerful tools for the basic researches and for differentiating ES and iPS cells into lineage-committed cells. Recently, we have developed an adenovirus (Ad) vector for efficient transduction into ES and iPS cells. We showed that Ad vectors containing the cytomegalovirus enhancer/β-actin promoter with β-actin intron (CA) promoter or the elongation factor (EF)-1α promoter were the appropriate for the transduction into ES and iPS cells. We also found that enforced expression of a PPARγ gene or a Runx2 gene into mouse ES and iPS cells by an optimized Ad vector markedly augmented the differentiation of adipocytes or osteoblasts, respectively. Thus, a gene transfer technique using an Ad vector could be an advantage for the regulation of stem cell differentiation and could be applied to regenerative medicine based on ES and iPS cells.

Adipocytes as a vehicle for ex vivo gene therapy: Novel replacement therapy for diabetes and other metabolic diseases.

Because of its availability and recent advances in cell biology, adipose tissue is now considered an ideal target site for the preparation of recipient cells and for the transplantation of gene-transduced cells for supplementation of therapeutic proteins. Inherited or acquired serum protein deficiencies are the ideal targets for gene therapy. However, to develop an effective ex vivo gene therapy-based protein replacement treatment, the requirements for the recipient cells are different from those for standard gene therapy that is intended to correct the function of the recipient cells themselves. To meet the requirements for such a therapeutic strategy, recent in vitro and animal model studies have developed new methods for the preparation, culture, expansion and manipulation of adipose cells using advanced gene transduction methods and transplantation scaffolds. In this short review, we introduce the progress made in novel adipose tissue-based therapeutic strategies for the treatment of protein deficiencies by our group and other investigators, and describe their future applications for diabetes and other metabolic diseases. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00133.x, 2011).

Construction of a high-performance human fetal liver-derived lentiviral cDNA library

The gene transduction method is a very powerful tool, not only in basic science but also in clinical medicine. Regenerative medicine is one field that has close connection with both basic and clinical. Recently, it has been reported that induced pluripotent stem (iPS) cells can be produced from somatic cells by a three or four gene transduction. We have also recently reported that lentiviral gene transfer of the tal1/scl gene can efficiently differentiate non-human primate common marmoset ES cells into hematopoietic cells without the support of stromal cells. In this study, we constructed a high-performance human fetal liver-derived lentiviral expression library, which contains a high number of individual clones, in order to develop a very helpful tool for understanding early hematopoiesis and/or hepatocytosis for future regenerative medicine. Our lentiviral cDNA library consisted of more than 8 x 10(7) individual clones, and their average insert size was >2 kb. DNA sequence analysis for each individual inserted cDNAs revealed that >60% contained the full-length protein-coding regions for many genes including cytokine receptors, cytoplasmic proteins, protein inhibitors, and nuclear factors. The transduction efficiency on 293T cells was 100% and the average size of an integrated cDNA was ~1.1 kb. These results suggest that our lentiviral human fetal liver cDNA expression library could be a very helpful tool for accelerating the discovery of novel genes that are involved in early hematopoiesis and hepatopoiesis and to make the use of iPS cells more efficient in the field of regenerative medicine.

Gene transduction by sonoporation

Gene transduction technologies are essential tools for understanding of gene functions or gene cascades underlying embryogenesis. In this review, we introduce a gene transduction method using microbubble and ultrasound (hereafter referred to as sonoporation). Sonoporation is carried out with relatively simple procedures and easily transduces genes into mesenchymal cells without significant damage to target tissues. Therefore, sonoporation is effective for gene transduction to study the molecular mechanisms of morphogenesis.

Sonoporation mediated transduction of pDNA/siRNA into joint synovium in vivo

The purpose of this study was to investigate the usefulness of sonoporation method on in vivo transduction of plasmid DNA (pDNA) and small interfering RNA (siRNA) into joint tissue. pGEG.GL3 plasmid was mixed with microbubble and injected into knee joints of rats. Ultrasound sonication was performed percutaneously. Three days after injection, GL3 expression of synovial tissue was determined by luciferase assay and RT-PCR. siRNA specific for GL3 (siGL3) or nonspecific siRNA were mixed with pGEG.GL3 plasmid and transduced by sonoporation. siRNA specific for EGFP (siEGFP) was transduced into the knee joints of EGFP transgenic rats, and gene silencing effects for endogenous gene were examined. To determine the localization of transduced siRNA, fluorescently labeled siRNA was transduced into joints. The expression of GL3 in the synovium was significantly enhanced by sonoporation. The gene expression was only seen in the synovium of the knee joint. The expression of GL3 was remarkably suppressed by co-transduction of siGL3, but not suppressed by nonspecific siRNA. siEGFP transduced by sonoporation attenuated green fluorescence on the surface layer of synovium of EGFP transgenic rats. The fluorescently labeled siRNA was seen in the synovium around the patella, femur, and tibia. Sonoporation is examined as a recent, novel, gene transduction method, and the advantage of this technique is minimal invasiveness. In this study, we showed that pDNA/siRNA can be transduced specifically into the joint synovium using sonoporation. The present method may be useful in nucleic acid therapy for joint disorders.

Efficient and controlled gene expression in mouse pancreatic islets by arterial delivery of tetracycline-inducible adenoviral vectors

Gene transfer with adenovirus vectors has been used extensively for pancreatic islet research. However, infection efficiency varies among reports. We reevaluated the infection efficiency, defined here as the percentage of islet cells expressing transgenes, in mouse islets. When the isolated islets were infected with adenoviruses, the infection efficiency was found to be 30-40% and the transduced cells were distributed in the islet periphery. Collagenase treatment of isolated islets before infection increased the infection efficiency to 70%, but with suppression of glucose-stimulated insulin secretion. To explore more efficient strategies, we employed arterial delivery of virus particles to islets in situ. Delivery of adenovirus (approximately 10(8) particles per pancreas) through the celiac and superior mesenteric arteries is highly efficient, resulting in more than 90% transduction without impairing glucose-stimulated insulin secretion. Arterial delivery of an adenovirus harboring glycerol kinase cDNA allowed us to observe glycerol-stimulated insulin secretion from mouse islets, which was not observed when we employed the conventional method. Furthermore, the arterial delivery method combined with a tetracycline-inducible adenovirus system induced efficient and controlled transgene expression. Our data provide new insights into gene transduction methods using recombinant adenoviruses in mouse islets, and are therefore anticipated to contribute to future basic and clinical islet research applications.

Repair of Articular Cartilage Defect by Intraarticular Administration of Basic Fibroblast Growth Factor Gene, Using Adeno-Associated Virus Vector

The objective of this study was to establish the potency of adeno-associated virus (AAV) as a viral vector to transport the basic fibroblast growth factor (bFGF) gene into synovial tissue, and to evaluate the consequent repair of articular cartilage defects. In the in vitro study, LacZ- and bFGF-encoding genes were transduced into rabbit synoviocytes by recombinant adeno-associated virus (AAV) vector, and the cells were cultured for 2 weeks. The percentage of successfully transduced LacZ-positive cells was assessed by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining, and the concentration of bFGF in the culture supernatant was confirmed by bFGF-specific enzyme-linked immunosorbent assay. In the in vivo study, 12- to 14-week-old Japanese white rabbits (all female) were used. AAV-bFGF was administered into an artificially created full-thickness defect (5 mm in diameter and 3 mm deep) in the patellar groove of the distal femur. Cartilage repair was subsequently monitored at 4, 8, and 12 weeks, by macroscopic and histological examination, and results were graded on the basis of semiquantitative scores. lacZ gene expression in synoviocytes reached more than 93% within the first 2 weeks, and the mean bFGF concentration in the culture supernatant of the bFGF gene-transduced group was significantly increased (p < 0.01). Semiquantitative macroscopic and histological assessment indicated that the average score was significantly better in the bFGF-transduced group throughout the observation period, suggesting better cartilage repair. These results demonstrate that gene transfer into synoviocytes, using the AAV vector, was a potent method of gene transduction. Moreover, after intraarticular administration of AAV-bFGF, constant expression of bFGF in the knee joints resulted in substantial cartilage regeneration that, with further long-term study, could possibly merit consideration for clinical application.

735. Femtosecond Laser- A New Intradermal DNA Delivery Method for Gene Expression and Vaccination

The skin is an ideal target for naked DNA delivery for gene expression and vaccination, due to ease of administration and presence of a large number of antigen-presenting cells within the tissue. There are several different methods to transfer naked DNA to the skin. However, some of these delivery methods encounter low level of transfection efficiency and lack of sustained expression. We propose a new and highly efficient method of dermal gene transduction - the Laser Beam Gene Transduction method (LBGT). Methods. Using a femtosecond infrared titanium sapphire laser beam as a laser source illumination, the shaved back skin of Balb/c mice was exposed for assessment of LBGT. The pCAG/Luc construct was used for luc expression. For in vivo real time continuous luc expression, the bioluminescence CCCD system was used (Zeira et al. Mol. Ther. 4:239, 2001). For assessment of this delivery method for vaccination, we used the pCMV/HBsAg vector. Results. A single intra-dermal injection of 10mg of pCAG/Luc followed by laser beam illumination for 2 minutes, resulted in an increase of transgene expression for up to an average of 1000-fold on day 43, relative to naked DNA injection. Characterization studies revealed the optimal conditions for LBGT into the mice dermis. Gene expression following LBGT continued for > 50 days. In mice immunized with the HBsAg plasmid, (10|[mu]|g) on day 0 and 21, HBsAg antibody titers were detected shortly after the first injection and continued to increase over one month, and were significantly higher than titers obtained by naked DNA administration without LBGT. Conclusion. Delivery of DNA plasmid into the dermis by LBGT is highly efficient for transgene expression and for elicitation of immune response. These results provide a proof of principle that femtosecond laser may appear as a novel method for the administration of DNA-based vaccines.

Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells by Their Genetic Manipulation Using the Third Generation Lentiviral Vector.

Since the successful establishment of human embryonic stem (ES) cell lines in 1998, transplantation of differentiated ES cells to specific organ has been expected to complete its defective function. For the realistic medicine, the preclinical studies using animal model systems including non-human primates are essential. We have already demonstrated that non-human primates of common marmosets (CM) are suitable for the laboratory animal models for preclinical studies of hematopoietic stem cell therapy. In this study, we investigated the in vitro and in vivo differentiation of CM ES cells to hematopoietic cells by exogenous gene transfer methods in order to study the feasibility of future gene modified ES cell therapy. First, we tried various in vitro culture conditions including systems using embryoid bodies or co-culturing with stromal cells to induce hematopoietic cells, but the frequency of inducing hematopoietic cells was very low. The expression of CD45 and gata1 could not be detected in both conditions, suggesting that our culture conditions were incomplete for induction of hematopoietic cells from CM ES cells. Next we examined gene transduction methods by using VSV-G pseudotyped human immunodeficiency virus (HIV) vectors. We constructed the HIV vectors containing hematopoietic genes such as tal1/scl, gata1, gata2, hoxB4 and Lh2 genes under the EF1a promoter and transduced them into CM ES cells. Only in the case of tal1/scl overexpression, not other genes, hematopoietic induction from CM ES cells was dramatically increased and multi-lineage blood cells consisting of erythroid cells, granulocytes, macrophages and megakaryocytes, were confirmed by immunochemical and morphological analyses. Furthermore, RT-PCR results showed that several hematopoietic marker genes including CD34 were expressed higher in the tal1/scl overexpressed ES-derived cells. After the xenotransplantation of ES-derived cells into the immunodeficient mice, CM CD45+ cells and immature erythroids and megakaryocytic cells were observed only in the ES-tal1-injected mice, indicating that enforced expression of tal1/scl into ES cells led to highly efficient hematopoietic cell differentiation in vivo. Taken together, it was suggested that the transduction of exogenous tal1/scl cDNA into ES cells by HIV vector was the promising method for the efficient differentiation from CM ES cells to hematopoietic stem cells. Further examinations are required to determine the long-term hematopoietic reconstitute capacity and the safety of the tal1/scl transduced ES cells in marmoset for the purpose of developing new hematopoietic stem cell therapy.

Femtosecond infrared laser—an efficient and safe in vivo gene delivery system for prolonged expression

The major advantages of “naked DNA gene therapy” are its simplicity and a low or negligible immune response. Gene delivery by DNA electroporation (EP) involves injection of DNA and the application of a brief electric pulse to enhance cellular permeability. Although EP is an efficient gene transduction technique in rodents, it requires much higher voltages (>500 V) in larger animals, and hence, in practice it would be hazardous for human patients, as it would cause serious tissue damage. To overcome the obstacles associated with EP-mediated gene delivery in vivo, we developed a new method of gene transduction that uses laser energy. The femtosecond infrared titanium sapphire laser beam was developed specifically for enhancing in vivo gene delivery without risks of tissue damage. System optimization revealed that injection of 10 μg naked DNA into the tibial muscle of mice followed by application of the laser beam for 5 s, focused to 2 mm depth upon an area of 95 × 95 μm2, resulted in the highest intensity and duration of gene expression with no histological or biochemical evidence of muscle damage. We assessed the potential clinical application of LBGT technology by using it to transfer the murine erythropoietin (mEpo) gene into mice. LBGT-mediated mEpo gene delivery resulted in elevated (>22%) hematocrit levels that were sustained for 8 weeks. Gene expression following LBGT was detected for >100 days. Hence, LBGT is a simple, safe, effective, and reproducible method for therapeutic gene delivery with significant clinical potential.

A retroviral vector system 'STITCH' in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T lymphocytes.

Genetic engineering of T lymphocytes for adoptive clinical immunotherapy calls for efficient gene transduction methods. Therefore, a transient retroviral gene transduction system 'STITCH' was developed comprising pSTITCH retroviral vector encoding the transgene, plasmids encoding Moloney murine leukemia virus gag/pol and gibbon ape leukemia virus envelope, and the human kidney cell line 293T as a packaging line. Cotransfection of retroviral vector and packaging plasmids in 293T cells results in the production of GALV env pseudotyped viral particles with a titer of 10(7) infectious units per milliliter. The 'STITCH' gene transduction system efficiently transduces genes into activated human T lymphocytes derived from healthy donors and cancer patients. The efficacy of gene transduction is donor-independent. A direct application of the 'STITCH' gene transduction system is the genetic engineering of activated human T lymphocytes to induce expression of antibody based chimeric receptors in their membrane. Introduction of these chimeric receptors into activated human T lymphocytes graft these cells with specificity for, for example, renal cell carcinoma. In order to study the effect of the chimeric receptor gene structure on the processes ultimately leading to functional membrane expression, we designed a number of different chimeric receptor gene structures and subsequently compared their membrane expression on 293T cells and activated human T lymphocytes. Distinct membrane expression densities were observed on 293T cells and human T lymphocytes for the different chimeric receptor gene constructs. Gene transduction of activated human T lymphocytes with four out of five chimeric receptor gene constructs resulted in functional expression of chimeric receptor as demonstrated by specific recognition and cytolysis of renal cell carcinoma.

Adenovirus-mediated gene transfer during initial organogenesis in the mammalian embryo is promoter-dependent and tissue-specific.

Replication-defective adenoviruses have received increasing attention as vectors for exogenous gene administration in a variety of experimental and pathological conditions. However, little information exists about their utility for in utero gene therapy, and no information exists concerning their efficacy for gene delivery during initial organogenesis in the mammalian embryo. To evaluate the feasibility of using these vectors for exogenous gene transduction during the initial stages of organogenesis in the mammal, we injected an adenovirus vector carrying the bacterial beta-galactosidase (lacZ) gene under the control of either the cytomegalovirus (CMV) promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) into early, post-gastrulation, mouse embryos, and evaluated expression following 36-48 h in culture. These studies suggest that adenovirus-mediated gene delivery may provide an efficient method of gene transduction during critical developmental stages with no detectable adverse effects on normal development during early morphogenesis. In addition, the type of promoter used had a significant effect on the tissue distribution of gene expression.