Generation of genetic modified pigs devoid of GGTA1 and expressing the human leukocyte antigen-G5 / 生物工程学报

Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.

Research progression on the first-line biological target therapy of advanced / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To develop a new type of CD7 chimeric antigen receptor modified T cell (CD7-CAR-T) for the treatment of CD7 positive acute myeloid leukemia (AML), and to observe its killing effect on CD7 positive AML cells. Methods: The CD7-CAR lentiviral vector was constructed based on the CD7 Nanobody sequence and costimulatory domain sequence of CD28 and 4-1BB. The lentiviral particles were packaged and used to co-transfect human T cells with protein expression blocker (PEBL), so as to prepare CD7- CAR-T cells. Real time cellular analysis (RTCA) was used to monitor the cytotoxicity of CD7-CAR-T cells on CD7 overexpressed 293T cells. Flow cytometry assay was used to detect the effect of CD7-CAR-T cells on proliferation and cytokine secretion of AML cells with high, medium and low CD7 expressions (KG-1, HEL and Kasumi-1 cells, respectively). Results: CD7-CAR-T cell was successfully constructed and its surface expression of CD7 was successfully blocked. Compared with T cells, CD7-CAR-T cells could significantly inhibit the proliferation of CD7-293T cells and promote the release of TNF, Granzyme B and INF-γ; in addition, CD7-CAR-T cells also significantly promoted the apoptosis (t=147.1, P<0.01; t=23.57, P<0.01) and cytokine release (P<0.05 or P<0.01) in CD7 positive KG-1 and HEL cells, but had little effect on Kasumi-1 cells that only expressed minimal CD7 antigen (t=0.7058, P>0.05). Conclusion: CD7-CAR-T cells can specifically kill CD7-positive AML cells in vitro.

Regulatory perspective for chemistry manufacturing and controls considerations in chimeric antigen receptor-modified T cell therapy / 药学学报

As a living cell product, chimeric antigen receptor (CAR)-T cell therapy displays multiple characteristics including the diversity of raw materials, the complexity of manufacturing process and the complementarity of quality control set. Pharmaceutical research and evaluation of CAR-T cell therapy are fundamentally different from small molecule and macromolecular recombinant proteins. Chemistry manufacturing and controls (CMC) review of investigational new drug (IND) submission for CAR-T therapy should especially pay attention to above unique characteristics and focus on potential risks to ensure clinical safety. Based on questions and concerns from recent CMC review practice and workshop on CAR-T cell therapy IND application, the critical points to consider for CMC study is proposed, and questions related to supplementation are also discussed in this review to accelerate the clinic translation of CAR-T therapy.

Advance of gene-modified non-human primate models / 生物工程学报

Non-human primates would be particularly valuable in life sciences and biomedical research area. Gene-modified monkeys with gene overexpression or loss of function have been successfully generated with the rapid advance in gene manipulation technology such as lentivirus infection and programmable nucleases (ZFN, TALEN, CRISPR-Cas9). Here we review the recent development on gene-modified monkey generation by lentivirus and programmable nucleases. Then we discuss three concerns, the long time for sexual maturation, the off target and the mosaicism of founders, which limit the wide application of gene-modified non-human-primates. At last, hotspots and future trend for gene-modified non-human-primates generation are proposed.

Changes of brain - derived neurotrophic factor expression in gene modified bone marrow mesenchymal stem cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To study the changes of brain - derived neurotrophic factor ( BDNF ) expression in gene modified bone marrow mesenchymal stem cells ( BMSC) . ●METHODS:BMSC were divided into blank control group ( without transfected BMSC ) , negative control group ( empty vector without BDNF gene transfected BMSC) and experimental group ( BDNF gene transfected BMSC) . The expression of BDNF mRNA in BMSC was measured by Realtime PCR, and the expression of BDNF in BMSC was measured by ELlSA. ●RESULTS:The BDNF mRNA expressions of 3, 4, 5, 6, 7 and 8-generation BMSC cells in the experimental group were higher than those in the blank control group and negative control group. The differences were statistically significant (P3: F=491. 788, P ●CONCLUSION:Long-term expression of BDNF in BMSC can be enhanced by genetic engineering.

Release of L-EK in cerebrospinal fluid induced by intrathecal HEK293 cells modified with human preproenkephalin gene and the analgesic efficacy of L-EK for bone cancer pain / 中华麻醉学杂志

Objective To evaluate the release of leucine-enkephalin (L-EK) in the cerebrospinal fluid induced by intrathecal human embryonic kidney (HEK293) cells modified with human preproenkephalin (hPPE)gene and the analgesic efficacy of L-EK for bone cancer pain.Methods Forty CIBP female Sprague-Dawley rats were randomized into transplantation (CIBP+ hPPE/HEK293,n =20) and control (CIBP + HEK293,n =20)groups using a random number table.At 1 day before inoculation of cancer cells (T1,baseline) and 8,15,21,25,32 and 35 days after inoculation (T2-7),thermal paw withdrawal latency (TWL) was measured,and the number of licking/biting the claw on the transplantated side and degree of hindlimb limping during free activities were recorded.After observation at T4,10 rats were chosen from each group and sacrificed and the cerebrospinal fluid of rats was collected in an ice bath for detection of hPPE expression using radioimmunoassay.Results Compared with control group,TWL was significantly prolonged,the concentration of L-EK in the cerebrospinal fluid was increased,and the number of licking/biting the claw on the transplantated side and degree of hindlimb limping during free activities were decreased at T4-7 in transplantation group.Conclusion Intrathecal HEK293 cells modified with hPPE gene can continuously secrete L-EK and mitigate bone cancer pain.

Scientific management of animal models of human diseases / 中华医学科研管理杂志

In recent years,many kinds of animal models of human diseases with different genetic background were developed.Some were established in China,others were imported from abroad.Precious were these animal models,their biological features could be lost for various reasons,for instance,reverse from mutation,lost of modified or constructed gene,etc.This study provided some suggestions for scientific management of the animal models to in terms of quarantine inspection,specific feeding method and methods to maintain gene stability,so as to preserve their biological features.

In vitro Observation on the Immunological Characters of IFN-? Induced, IL-2 Gene-Modified Human Primary Fibroblasts / 中国肿瘤生物治疗杂志

It was an important strategy in cancer gene therapy that transferring foreign gene to fibroblasts to express synthetic protein to exert antitumor effect. But most of studies regarded the foreign gene-modified fibroblasts as a by-stander, only a delivery source of synthetic protein, and neglected their irnmunological characters. In this paper, human primary dermal fibroblasts were modified by human IL-2 gene by retrovirus vector and induced by human IFN-?. The results showed that the MHC- I , MHC- II and CD40 expression on the surface of IFN-? induced fibroblasts were up-regulated significantly in comparison with those of non-induced cells. The IL-2, IL-1, IL-6 secretion were detected as a increased level in supematants of the IFN-?-induced, IL-2 gene modified fibroblasts. Because these molecules and cytokines play important roles in antigen presentation and effector activation, it can be inferred that the IFN-y induced, IL-2 gene modified fibroblasts could be used as antigen presenting cells in addition to delivery cells to activated the effector cells.