Chemistry, manufacturing and control considerations of rAAV-based gene therapy products / 药学学报

Recombinant adeno-associated virus (rAAV)-based vector has shown great promise for human gene therapy, due to its advantage in eliciting long-term transgene expression, absence of adverse effect, infection ability to both dividing and non-dividing cells, non-genomic integration, and low immunotoxity. To date, three AAV-based products have been authorized to enter European and American markets, and more than 200 rAAV-based candidates are in the process of clinic trails. Nevertheless, domestic industry is facing the challenge of manufacturing clinical grade rAAV vector, and regulatory agencies are lack of practical experience in assessing such products. Herein, this paper summarizes the latest research progress of rAAV-based gene therapy products, and discusses some quality assessment concerns in raw materials, manufacturing process and quality control, expecting to promote its clinical transformation and application.

Effect of Graft Composition on Acute Graft-Versus-Host Disease in Aplastic Anemia after Haploidentical Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between acute graft-versus-host disease and graft composition in patients with aplastic anemia(AA) after haploidentical hematopoietic stem cell transplantation.</p><p><b>METHODS</b>Fifty-seven cases of AA after haploidentical hematopoietic stem cell transplantation were retrospectively analyzed. All the patients were divided into 2 groups according to whether presence or absence grade Ⅱ-Ⅳ aGVHD, the relationship between aGVHD and graft composition was analyzed by comparing the differences of graft components between the 2 groups.</p><p><b>RESULTS</b>Fourteen out of 57 patients had grade Ⅱ-Ⅳ aGVHD and the other 43 did not have grade Ⅱ-Ⅳ aGVHD. The mononuclear cells, CD3, CD4, CD8, NK cells, NKT cells, B cells and Treg cells were not significantly different between the 2 groups (P>0.05), the CD34 cell count in the patients with grade Ⅱ-Ⅳ aGVHD was 3.85（1.73-10.61）×10/kg, which was significantly lower than that without grade Ⅱ-ⅣaGVHD: 6.31（2.98-19.35）×10/kg (P<0.05).</p><p><b>CONCLUSIONS</b>The incidence of grade Ⅱ-Ⅳ aGVHD may be related with CD34 cell count in AA after haploidentical hematopoietic stem cell transplantation..</p>

Efficacy and Safety of Co-transplantation of Haploidentical-HSC with Umbilical Cord Mesenchymal Stem Cell in Children with Hematologic Malignancies / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyse the efficacy and safety of co-transplantation of umbilical cord mesenchymal stem cell(UC-MSC) with haploidentical hematopoietic stem cell transplantation(hi-HSCT) in children with hematologic malignancy.</p><p><b>METHODS</b>The clinical data of 47 children undergoing hi-HSCT were retrospectively analyzed from November 2003 to November 2014, among them 34 patients received UC-MSC from October 2011 to November 2014, and another 13 patients without UC-MSC from November 2003 to September 2011. The median follow-up time was 20(0.5-67) months.</p><p><b>RESULTS</b>No adverse events were observed after the UC-MSC transplantation. The engraftment rate, the median neutrophils engraftment time and platelet engraftment time all were not significantly different between hi-HSCT and hi-HSCT+UC-MSCT(P>0.05). The three-years cumulative overall survival (70.6% vs 23.1%),(P=0.004), three-years cumulative disease-free survival(52.9% vs 0) (P=0), and early cytomegalovirus (CMV) viremia (91.2% vs 38.5%) (P=0) in UC-MSC+hi-HSCT group were statistically significantly higher than that in the conventional hi-HSCT group. The morbidity of aGVHD (44.1% vs 92.3%) (P=0.003), I-II aGVHD (26.5% vs 61.5%) (P=0.041) and transplantation-related mortality (11.8% vs 46.2%) (P=0.017) in UC-MSC+hi-HSCT group was statistically significantly lower than that in hi-HSCT group, however, the morbidity of III-IV aGVHD (17.6% vs 30.8%), cGVHD (26.5% vs 30.8%), HC (35.3% vs 7.7%), pulmonary infection (52.9% vs 46.2%) and relapse rate (32.4% vs 53.8%) were not statistically significantly different (P>0.05) between the 2 groups.</p><p><b>CONCLUSION</b>The application of umbilical cord mesenchymal stem cell in children undergoing hi-HSCT is safe, the UC-MSC can improve the overall survival, disease-free survival and reduce transplantation-related mortality. UC-MSC can reduce the morbidity of aGVHD, but increase the early infection of CMV, however it is nothing for the pulmonary infection and relapse in the children after hi-HSCT.</p>

Clinical Observation of Haploidentical Hematopoietic Stem Cell Transplantation Combined with Bone-marrow Derived Mesenchymal Stem Cells Transfusion for Treatment of Children with Severe Aplastic Anemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the efficacy and safety of haploidentical hematopoietic stem cell transplantation(hi-HSCT) combined with bone-marrow derived mesenchymal stem cell (BM-MSC) tranfusion for treatment of children with severe apastic anemia(SAA).</p><p><b>METHODS</b>The clinical data of 25 children with SAA undergoing hi-HSCT and BM-MSC tranfusion were retrospectively analyzed from August 2014 to July 2016.</p><p><b>RESULTS</b>neutrophil engraftment was achieved in all 25(100%) children, with the median time 12(11-22) days. The median time of platelet engraftment was 21(11-130) days in 23(92%) children. Acute graft-versus-host disease(aGVHD) was observed in 16(64%) cases, including 11 case of grade I and 5 cases of aGVHD grade II-IV, and one of them died of grade IV of skin, gut and liver at day 115; 5 cases of chronic GVHD were found, all of them were limited cGVHD. Cytomegalovirus (CMV) viremia was observed in 23(92%) cases, but no CMV disease was developed after therapy. 3 cases of post-transplant lymphoroliferative disease with 23 EBV viremia positive occurred, all of them were cured after rituximab. Hemorrhagic cystitis appeared in 9 cases with only one case of grade III, 22 children suffered from infection, involving 10 cases in lung and 4 cases in liver, 1 patient was diagnosed as Guillain-Barre syndrome. Autoimmune hemolytic anemia was recorded in 1 patient, 22 children survived during a median following-up time of 14(3-27) months.</p><p><b>CONCLUSION</b>The hi-HSCT combined with BM-MSC transfusion for treatment of children with SAA has been confirmed to be safe and feasible.</p>

Clinical Study on Treatment of Juvenile Myelomonocytic Leukemia with Haploidentical-Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the therapeutic efficacy of haploidentical-hematopoietic stem cell transplantation (hi-HSCT) for patients with juvenile myelomonocytic leukemia (JMML).</p><p><b>METHODS</b>The engraftment of hematopoietic stem cells, incidence of graft versus host disease (GVHD), infection, relapse, and survival of 6 JMML patients received hi-HSCT were retrospectively analyzed.</p><p><b>RESULTS</b>Six (6 males) JMML patients received hi-HSCT from haplo-HLA-matched related donors. The results showed that the hematopoictic stem cells in all 6 patients were grafted successfully. Two cases of JMML died of pulmenary infections, other 4 cases survive without disease. Acute GVHD occurred in 3 patients and chronic GVHD occurred in 1 patients. The overall survival, disease free survival and relapse rates were 66.7%, 66.7%, 0%, respectively.</p><p><b>CONCLUSION</b>The hi-HSCT is an effective method for treatment of patients with JMML, but there also is a serial problems to be resolved.</p>

Mesenchymal Stem Cells Combined with Budesonide, Almeterol and Azithromycin for the Treatment of Bronchiolitis Obliterans Syndrome after Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate the safety and effectiveness of a novel therapeutic regimen for bronchiolitis obliterans sydrome (BOS) affter hematopoietic stem cell transplantation (HSCT).</p><p><b>METHODS</b>Seven patients who had received HSCT and had been diagnosed as BOS were enrolled in this study. They received weekly intravenous injection of umbilical cord-derived mesenchymal stem cells (MSC) at a dose of 1 × 10(6)/kg for 4 weeks. Budesonide was given orally at a daily dose of 0.25 g, and salmeterol was inhaled at a dose of 4.5 µg for 3 times per day. Methylprednisolone was given at a dose of 1 mg/(kg·d) for 2 weeks when respiratory failure occured. The dose of methylprednisolone was tapered to 0.25 mg/(kg·d) after 4 weeks and was adjusted according to the occurrence and severity of chronic graft-versus-host disease (cGVHD).</p><p><b>RESULTS</b>The therapy was generally safe and no severe acute toxicity was observed. One patient died of heart failure during the treatment, the other 6 patients were alive and the pulmonary function parameters including FEV1, FEV1/FVC, PaO2 and AaDO2 were significantly improved after 6 months as compared with the baseline parameters (P < 0.05).</p><p><b>CONCLUSION</b>MSC combined with budesonide, almeterol and azithromycin has been confirmed to be generally safe and can reduce the dose of glucocorticoid in treatment of BOS after HSCT.</p>

Analysis of Risk Factors and Therapeutic Strategies for Relapse of Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the risk factors of patients with relapsed leukemia after allogeneic hematopoietic stem cell transplantation, and to explore the therapeutic strategies for recurrence.</p><p><b>METHODS</b>The Cox proportional hazard regression model was used for univariate and multivariate analysis of transplantation-related index, a single center retrospective study of clinical data of 202 cases of leukemia received allo-HSCT from March 2004 to October 2014 had been conducted to screen the risk factors for recurrence after transplantation.</p><p><b>RESULTS</b>In the leukemia patients received allo-HSCT, 68 cases relapsed. The relapse rate was 33.6%. The median time of relapse was 4(1.5-26 ) months. Univariate analysis indicated that there were 5 risk factors related with the disease relapse(P<0.05), including the type of disease, extramedullary disease prior to transplant, the course of induced remission, the status of disease at HSCT and chronic graft versus host disease(cGVHD). Multivariate analysis showed that extramedullary disease prior to transplant(RR=2.622, 95%CI 1.139-6.037), the course of induced remission(RR=1.156, 95%CI 0.682-1.957), cGVHD (RR=1.728,95%CI 0.999-2.991) were independent risk factors for relapse of the patients received transplantation. Treatment strategies for the relapsed patients included withdraw immunosuppressant, donor lymphocyte infusion, systemic chemotherapy and local radiotherapy, targeted therapy, and second transplantation. Individualized choice was needed according to the relapsed site. The relapse-related mortality was 25.2%.</p><p><b>CONCLUSION</b>The relapsed patients with leukemia after allo-HSCT have poor prognosis, early interference has good effect. The evaluation and prevention of risk factors before transplantation is even more important.</p>

Delayed Hepatic Veno-Occlusive Disease after Haploidentical Hematopoietic Stem Cell Transplantation:A Report of Six Cases / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate the morbidity, risk factors, clinical characterisitics, treatments and prognosis of delayed hepatic veno-occlusive disease(HVOD) after haploidentical hematopoietic stem cell transplantation (hi-HSCT).</p><p><b>METHODS</b>The clinical data of 208 patients undergoing hi-HSCT were retrospectively analyzed.</p><p><b>RESULTS</b>Six patients were diagnosed with delayed VOD, among them 4 patients were moderate VOD and 2 patients were severe VOD. The incidence of VOD after hi-HSCT was 2.88%, the median onset time was 44.5(30-57) days after transplant, 2 patients died of multiple organ failure (MOF) due to rapid progress of disease. With intravenous administration of defibrotide, 4 patients displayed encouraging response, but 2 patients died of hepatic acute graft-versus-host disease (aGVHD), 1 had bone marrow relapse and the other one was cured.</p><p><b>CONCLUSION</b>Norethindrone is one of the high risk factors, while sex, age and disease status are irrelevant to the occurrence of VOD. Unfractionated heparin (UH) can effectively decrease the morbidity. Pretransplant hepatic function reserve, high dose preconditioning regimens and pharmacotherapy may result in delayed VOD onset. The delayed VOD has the same clinical features and treatment-response as early VOD, but a poorer prognosis is usually observed. A larger amount of samples (patients) is needed to research the relationship of the delayed VOD with hi-HSCT. Defibrotide can effectively increase the survival rate of VOD patients.</p>

Clinical Analysis of Cytomegalovirus Infection after Different Patterns of Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical difference of cytomegalovirus (CMV) infection between HLA-matched allogeneic hematopoietic stem cell transplantation (allo-HSCT) and haploidentical hematopoietic stem cell transplantation (hi-HSCT).</p><p><b>METHODS</b>The clinical data of 83 patients who had undergone allo-HSCT were retrospectively analyzed. Out of them 50 patients underwent hi-HSCT and 33 patients received grafts from HLA-matched donors. The sera of all recipients and donors were CMV-negative before transplantation. All patients accepted myeloablative regimen without total body irradiation. PCR was performed to detect CMV in the peripheral blood twice a week after neutrophil recovery. CMV-DNA>500 copies/ml was defined as CMV viremia.</p><p><b>RESULTS</b>68 patients (81.9%) were diagnosed as CMV viremia before 100 days after transplantation. The incidence of CMV infection in hi-HSCT group was 90% and significantly higher than that in HLA-matched HSCT group (69.7%) (P < 0.05). All the patients responded well to anti-CMV therapy; however, 63 cases experienced CMV reactivation. The occurrence rate of CMV reactivation in hi-HSCT group (95.6%) was comparable to that in HLA-matched HSCT group (87.0%) (P > 0.05). Univariate analysis showed that the transplantation pattern, the recovery time of peripheral neutrophils and the occurrence of acute graft-versus-host disease (aGVHD) significantly related to the episode of CMV viremia, while the sex and age of the recipients, and the recovery time of platelets did not associate with the incidence. Further analysis found that the recovery time of neutropils and platelets in HLA-matched HSCT group were greatly shorter than those in hi-HSCT group (P < 0.05). The incidence of aGVHD was comparable between two groups however, incidence of severe aGVHD was significantly higher in hi-HSCT (P < 0.05).</p><p><b>CONCLUSION</b>The hi-HSCT is more susceptible to CMV infection, which may be related to the higher incidence of severe aGVHD and the relative delay of hematopoietic reconstruction as compared with HLA-matched HSCT.</p>

Allogeneic hematopoietic stem cell transplantation for chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia / 中国实验血液学杂志

This study was purposed to explore the therapeutic efficacy and influencing factors of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with chronic myelomonocytic leukemia (CMML) and in patients with juvenile myelomonocytic leukemia (JMML). The clinical data of 3 cases of CMML and 2 cases of JMML underwent allo-HSCT were analysed in term of multiparameter. The results showed that the hematopoietic stem cells in 5 patients grafted successfully. One case of JMML died of pulmonary disease, other 4 cases survive without disease. The analysis found that the disease burden before transplant, chromosome karyotype, acute GVHD II-IV and poor risk cytogenetics all associated with the relapse rate and disease-free survival rate of CMML. The low intensity conditioning regimen was better than myeloablative conditioning regimen. Type of donor and source of stem cells did not statistically and significantly affect OS and RFS. The splenectomy before allo-HSCT as well as spleen size at time of the alloHSCT did not influence on posttransplantation outcome of JMML. However, cord blood HSCT for JMML patients delayed hematologic recovery as compared to that of bone marrow or peripheral blood HSCT. The age, GVHD, HbF level played an important role in leukemia replace. It is concluded that the allogeneic hematopoietic stem cell transplantation is a curative regimen for CMML and JMML, but there also is a serial problems to be resolved.

Effect of umbilical cord MSC infusion on the pulmonary infection in haploidentical hematopoietic stem cell transplantation / 中国实验血液学杂志

This study was purposed to investigate the effect of umbilical cord mesenchymal cells (UC-MSC) infusion on the pulmonary infection in haploidentical hematopoietic stem cell transplantation (hi-HSCT). The infection of 83 patients underwent hi-HSCT was detected and analysed, among them 42 patients received haploidentical hi-HSCT, 41 received hi-HSCT combined with UC-MSC infusion. The results showed that 31 cases (73.81% ± 6.78%) were infected by cytomegalovirus and 21 cases in patients received hi-HSCT experienced pulmonary infections, including infections of fungal, virus, bacteria, tubercle bacillus, PCP and so on, the incidence rate was (50 ± 7.72)%; the infection of cytomegalovirus (CMV) was found in 31 cases, the incidence rate was (78.05 ± 6.46)%. In patients received hi-HSCT combined with UC-MSC, only 15 patients experienced pulmonary infection, the incidence rate was (36.59 ± 7.52)%, and the infection of cytomegalovirus (CMV) was observed in 32 patients, the incidence rate was (78.05 ± 6.46)%. There was no obvious statistical difference between two groups(P > 0.05). It is concluded that the UC-MSC infusion not increases the infection rate in hi-HSCT.

Analysis of CD4(+) T cell function in patients suffered from pulmonary infection in the early phase after haploidentical hematopoietic stem cell transplantation / 中国实验血液学杂志

This study was purposed to investigate the immune state of the patients suffered from pulmonary infection within 6 months after haploidentical hematopoietic stem cell transplantation (hi-HSCT). Adenosine triphosphate (ATP) value in CD4(+) T cells was measured by ImmuKnow method to assess the function of the lymphocytes in peripheral blood of 25 patients at 6 months after hi-HSCT. The results showed that the ATP level in CD4(+) T cells of the patients suffered from pulmonary infection was (179.88 ± 65.41) ng/ml before transplantation, (172.69 ± 118.81) ng/ml at 1 month, (218.15 ± 124.26) ng/ml at 3 months, (313.42 ± 116.29) ng/ml at 6 months after transplantation. The ATP level in CD4(+) T cells of the patients without pulmonary infection was (210.44 ± 94.71) ng/ml before transplantation, and decreased to (193.66 ± 133.69) ng/ml at 1 month and increased gradually to (355.02 ± 43.38) ng/ml at 3 months, (355.73 ± 93.85) ng/ml at 6 months after transplantation. It is concluded that the low ATP value in CD4(+) T cells in patients prior and post hi-HSCT may suggest probability of occurrence for infections, ATP value in CD4(+) T cells may be used as a reference indicator for clinical empirical use of antibiotics.

Rac1 expression and its effects on the cell cycle progression and apoptosis in human acute leukemic cell line HL-60 / 中国实验血液学杂志

The study was aimed to investigate the expression of Rac1 in human acute leukemic cell line HL-60 and effect of Rac1 on cell cycle progression and apoptosis. The mRNA expression of Rac1 in HL-60 cell line and normal human peripheral blood mononuclear cells (PBMNC) were examined by semi-quantitative RT-PCR. After transfection of HL-60 cells with different concentrations of Rac1 antisense oligodeoxynucleotide (ASODN) by means of FuGENE6, the survival, cell cycle, apoptosis of HL-60 cells were observed through MTT assay, FCM test, Wright-Giemsa, acridine orange/ethidium bromide (AO/EB) and Annexin V-FITC/PI staining test respectively. The results showed that Rac1 relative amount in HL-60 was 0.84 +/- 0.13, while it in the normal PBMNC was 0.26 +/- 0.1 (P < 0.01); the expression of Rac1 in HL-60 cells was significantly upregulated. Compared with sense oligodeoxynucleotide (SODN), HL-60 cell viability, after exposure to ASODN at a concentration of 2.0 g/L decreased, (73.7 +/- 5.0)% vs (93.2 +/- 3.0)% (P < 0.01), while the proportion of G(1) cells increased as (52.1 +/- 6.8)% vs (31.6 +/- 4.7)% (P < 0.05), the percentage of Annexin V positive cells increased, (19.2 +/- 2.1)% vs (4.1 +/- 1.7)% (P < 0.01), and HL-60 cells were observed to have formation of apoptotic bodies. The data presented indicate that Rac1 may be involved in regulation of HL-60 cell cycle and apoptosis, promote overproliferation of HL-60 cells and inhibit their apoptosis.

Inhibition of proliferation and induction of apoptosis and differentiation of leukemic cell line HL-60 by sodium valproate / 中国实验血液学杂志

To investigate the influence of sodium valproate (VPA) on proliferation, apoptosis and differentiation of HL-60 cell line, effect of VPA in various concentrations on proliferation of HL-60 cells was detected by MMT; Wright-Giemsa staining was performed to observe the morphologic changes of HL-60 cells; NBT experiment was used to test the differentiation of HL-60 cells; flow cytometry was used to observe cell cycles and analyze the apoptosis. The results indicated that the changes of the growth curve showed inhibition of proliferation of HL-60 cells. After a 24-48 hours culture with 2 mmol/L VPA, the cells exhibited nuclear shrinkage, pyknosis fragmentation and appearance of apoptosis bodies. The percentage of the annexin V(+)/PI(-) cells which were apoptotic increased from 2.9% to 17.1%; hypodiploid peak was observed; the percentage of HL-60 cells in G(1) phase increased from 51.1% to 84.6% and the cells in S phase decreased from 37.9% to 14.4%. After a week culture with 0.25 mmol/L VPA, the cells exhibited characteristics of differentiation. The percentage of NBT positive cells was (47 +/- 2)%. It is concluded that VPA can inhibit the proliferation of HL-60 cells while inducing differentiation and apoptosis of these cells. The mechanism needs to be further studied.

Difference of gene expression profiles between HL-60/VCR and HL-60 cells detected by human genome genechip / 中国实验血液学杂志

This study was aimed to detect the gene expression profile changes between human acute leukemia cell line HL-60 and VCR-resistance HL-60, and to investigate the underlying mechanisms of MDR by using genechip technology. In experiments, mRNA were harvested using TrizoL reagent from these two cell lines, through RT-PCR, the biotinylated nucleotide were incorporated into the cRNA during the in vitro transcription reaction. The high quality RNA was hybridized to the gene expression array--human genome U133A developed by Affymetrix. It was scanned by G2500A GeneArray Scanner and the acquired image was analysed by a series of softwares. The results showed that 5,507 genes were differentially expressed between human acute leukemia cell line HL-60 and VCR-resistant HL-60. Compared with HL-60, 3,100 genes were up-regulated and 2,407 genes were down-regulated in VCR-resistant cell line. These genes were involved in different cell activities such as growth regulation and signal transduction. Among the genes with remarkable differential expression between the two cell lines, 435 were up-regulated and 605 were down-regulated. It is concluded that many different kinds of genes are involved in the mechanism of MDR and there is an intricate molecular network that controls the sensitivity of leukemia cells to the chemotherapeutic agents. Genechip is an efficient tool for parallel gene expression analysis.