Preliminary Clinical Study For Treatment Of Duchenne Muscular Dystrophy With HLA Haploidentical Hematopoietic Stem Cell Transplantation

Background And ObjectiveDuchenne Muscular Dystrophy(DMD) is a fatal X-linked recessive muscular dystrophy.This disorder is characterized by progressive of proximal limbs muscle atrophy weakness and gastrocnemius pseudohypertrophy.DMD is one of the most common neuromuscular diseases and occurs in approximately one of3500male live births.DMD occurs as a result of mutations in the dystrophin gene (DMD,locus Xp21.2-21.3).The DMD gene encodes a427kDa cytoskeleton protein termed dystrophin,which can sustain the stability of sarcolemma and prevent the damage of various mechanical factors in muscle contraction. Nonsense mutation and frameshift mutation of DMD gene generate the absolute absence or nonfunctional dystrophin.The anomaly of this cytoskeleton protein lead to muscle fiber degeneration. This pathological changes in the muscle fiber is pathognomonic of DMD.Typically,DMD patients are clinically normal at birth although serum levels of the muscle isoform of creatine kinase are elevated as a consequence of muscle fiber degeneration. Initial physical signs such as muscle weakness are not generally observed until the child reaches2-3years of age. Confirmatory diagnosis is made up of genetic analysis and/or dystrophin detection. What is clinically evident in patients aged between of3and5years is waddling gait, difficulties in rising from the floor and climbing stairs. Subsequently follows pseudohypertrophy of the calf muscles, proximal limbs muscle weakness,and a positive Gowers’ sign. Wheelchair dependence is associated with progressive scoliosis. Progressive muscle wasting continues throughout life, resulting in loss of ambulation at about9-13years of age and the involvement of cardiac muscles and respiratory muscles. Death usually occurs in the late teens or early20s,from respiratory and/or cardiac complications.Currently, DMD still remains incurable, although20years has passed since the DMD gene and pathomechanism was identified. Pharmacological approaches and myoblast transplantation therapy are of limited effect while gene therapy and iPS (induced pluripotent stem cells) therapy have existential risks of cell mutation and activating oncogenes. DMD patients suffer from eternal agony both physically and emotionally,and this progressive muscle wasting disease burdens their family and social with financial and psychological.Trans-differentiation of stem cells provides the theoretic basis for therapy of hematopoietic stem cell transplantation (HSCT). The successful results of experimentation on animals and clinical practice of a few cases provide solid foundation for the application of allogeneic HSCT (Allo-HSCT) in the treatment of DMD,which give fresh hope to cure of DMD. HLA haploidentical hematopoietic stem cell transplantation (haplo-HSCT) solve the dilemma of deficiency of HLA identical donors.But compared with HLA matched hematopoietic stem cell transplantation,haplo-HSCT has high risks of graft failure,slow hematopoietic reconstitution, serious graft-versus-host disease (GVHD), delay of immune reconstruction, and increased incidence of fatal infections, high transplant-related mortality.In recent years,with the development of strategies in HSCT, the limitations associated with transplant across the MHC barrier were overcome, which makes haplo-HSCT a feasible therapeutic approach. After obtaining ethical approval from the Ethics Committee Board of the participating hospital for our study’s methods and conduct, we carry out preliminary clinical research for treatment of Duchenne muscular dystrophy with HLA haploidentical hematopoietic stem cell transplantation.Methods1.1Patient and donor eligibilityThe main subjects of this research are DMD patients that definitely diagnosed by genetic analysis and/or dystrophin detection. Selection criteria include:4years<age<16years, with normal hepatorenal function (exclusion of liver enzymes and serum creatinine abnormality due to DMD).,and Left Ventricular Ejection Fractions (LVEF)>40%.Patients and their legal guardians should fully understand the potential risk of this therapy before they apply for participation in treatment and sign the informed consent for approval from the Ethics Committee Board.Patients’ healthy lineal relatives are chosen as potential donors whose HLA typing are tested with Sequenceing-based typing (SBT).Favored donors are required have at least5/10of their HLA typing matched with the recipients.1.2Transplantation protocolWe conducted autologous hematopoietic stem cell mobilization,harvest and cryopreservation,to ensure safety of the patients. FBCA conditioning Regimen was used in the4patients:fludarabine [Flu30mg/m2/dx5d(-9,-8,-7,-6,-5d)], Busulfex [BU0.8mg/kg/q6h/dx4d(-8,-7,-6,-5d)], cyclophosphamide [CY60mg/kg/dx2d (-3,-2d)], anthymocyte globulin [ATG2.5mg/kg/d×5d (-5,-4,-3,-2, -1d)].Donors’ peripheral blood stem cells(PBSC) and/or bone marrow (BM) stem cells are collected after mobilization with granulocyte colony-stimulating factor (G-CSF). Fresh and unmanipulated BM and PBSCs are infused into the recipients in30minutes after harvest. GVHD prophylaxis is cyclosporine A (CsA,3mg/kg/d)+short-term MTX (15mg/m2iv on day+1and10mg/m2on days+3,+6and+11) Prostaglandin E1and heparin sodium are used in prevention of hepatic veno-occlusive disease (HVOD). Hyperhydration, diuresis, alkalinizing, mensa are given for for a prevention of hemorrhagic cystitis (HC). Ganciclovir is administered for prophylaxis of cytomegalovirus (CMV) infection and anti-infective drugs for prophylaxis.G-CSF and thrombopoietin (TPO) are administered to all recipients from day1after transplantation until hematopoietic recovery is realized.1.3Engraftment and chimerism monitoringBlood routine are monitored for information about haematopoietic reconstruction after HSCT. Hematopoietic chimerism was evaluated by fluorescence in situ hybridization (FISH) for sex-mismatched patient-donor pairs and by polymerase chain reaction with short tandem repeat (STR-PCR) for sex-matched pairs using peripheral blood samples from the donor and the recipient.1.4Protopathy follow-upSerum creatine kinase are monitored weekly,and selective tests of DMD gene are conducted using peripheral blood samples from post-transplantation DMD patients to gain information about gene correction. Limb motor function, myodynamia and nervous reflex were examined and assessed during follow-up.Results1.14patients met inclusion criteria4DMD male patients participate in research whose median age is9years (range:6-16years). The results of DMD gene mutations detection of the4patients were Exon51deletion,c.2804-1G>T mutation,Exon2-44deletion,E6c.436C>T mutation respectively.All4patients and their donors were haploidentical.In turn,HLA sequencing were5/10matched (mother),7/10matched (father),6/10matched (mother),5/10(mother). There is ABO blood type incompatibility between patient3and his donor (the donor is type A while the recipient is type O)1.2mononuclear cell counts transplantedThe median infused doses of mononuclear cell (MNC) in our4patients was8.19×106/kg.Patient1was infused twice PBSC and once BM stem cells from the donor altogether with a MNC total of6.68×108/kg. Patient2received both PBSC and BM stem cells transplant once with a MNC total of was6.68×108/kg. Patient3and case4only infused once PBSC transplant and their MNC counts were9.7×108/kg and5.56×108/kg (CD34+:2.45×106/kg) respectively.1.3EngraftmentAll4patients achieved hematopoietic reconstitution successfully. The median time to reach an absolute neutrophil count above0.5×109/L was+11days (range:+10d～+13d). The median time for platelet to recover to20×109/L was+10.5days (range+10d～+13d) and hemoglobin to90g/L needed a median time of+12.5days (range:+12～+28d)All the Laboratory test results showed absolute stable donor chimerism in the4post-transplant patients. STR-PCR test on+14d and FISH tests on+83d,+343d,+507d exhibited complete donor chimerism.The same result appeared in patient2on+13d by STR-PCR test and patient3on+15d by FISH test and patient4on+15d+87d by FISH test. Blood group of patient3was changed from A to O after transplant. It was detected that deficiency of the enzyme glucose-6-phosphate-dehydrogenase (G6PD deficiency) was combined in patient1was corrected after transplantation. 1.4Transplant-related complicationsAll4patients developed grade I acute GVHD (aGVHD) in a median time of+23d (range:+15d-+33d).2developed grade I chronic GVHD (cGVHD) in a median time of10.5d (+108-+113d).After administration of glucocorticoids and CD25monoclonal antibody,infusion of mesenchymal stem cells (MSC), aGVHD and cGVHD were successfully controlled. Follow-up of+654d,+338d,+325d,+275d post-transplant,GVHD symptoms was not observed in them. Patient1and patient4developed HC on+30d and+27d respectively.3of them developed oral mucositis (OM) at a median time of+4d (-11-+17d).Only patient4developed shingles on+194d. All patients experienced infectious complications included pulmonary infection for3and intestinal infection for1. Nevertheless, severe infections and fatal complications were not happening in them after treatment.4patients were all in good conditions and their life was not significantly declining compared with pre-transplant in the follow up..1.5Protopathy changesAll4patients’creatine phosphokinase levels decreased compared with that of pre-transplant and gene disorders correction were confirmed by tests of peripheral blood in post-transplant patients. Limb motor function droped a little in their first+50d post-transplant then improved on about+150d. As time went on, Gastrocnemius became softer and limb motor function recoveed to the level that of pre-transplant. Follow-up of+654d,+338d,+325d,+275d post-transplant, motor function of them stably maintains the level that of pre-transplant. All4patients in our research showed no clinical deterioration like other Duchenne muscular dystrophy patients.Conclusion1. HLA haploidentical hematopoietic stem cell transplantation can be used for the cure of Duchenne muscular dystrophy though risks did exist.2. FBCA conditioning regimen can ensure the successful engraftment of allogeneic hematopoietic stem cell. GVHD Prophylaxis of cyclosporine A+short-term MTX effectively prevents the development of severe GVHD after haplo-HSCT.3. Creatine phosphokinase levels decreased and gene disorders were corrected after haplo-HSCT in the4DMD patients.Limb motor function of them was improved to varying degrees in recent follow-up, but long-term outcomes need further follow-up and assessment.