title: Encapsulated human primary myoblasts deliver functional hFIX in hemophilic mice.
abstract: BACKGROUND
  Hemophilia B is a bleeding disorder caused by defective factor IX (FIX), currently treated by regular infusions of plasma-derived or recombinant FIX. We propose a gene therapy strategy based on the implantation of cells secreting FIX enclosed in alginate microcapsules as a highly desirable alternative treatment. We have reported sustained delivery of human factor IX (hFIX) in immunocompetent mice implanted with encapsulated primary mouse myoblasts engineered to secrete hFIX. As a step towards the treatment of human patients, in this study we report the implantation of encapsulated human primary myoblasts secreting hFIX in hemophilia B mice.
  
  
  METHODS
  Human primary myoblasts were transfected with plasmids pKL4M-hFIX, pLNM-betaIXL, pMFG-hFIX, and transduced with retrovirus MFG-hFIX. Two human primary myoblast clones secreting approximately 1 microg hFIX/10(6) cells/day were enclosed in biocompatible alginate microcapsules and implanted intraperitoneally into SCID and hemophilic mice.
  
  
  RESULTS
  Circulating hFIX (peak of approximately 120 ng/ml) was detected in hemophilia B mice on day 1 after implantation. Human FIX delivery was transient, however, becoming undetectable on day 14. Concurrently, anti-hFIX antibodies were detected. At the same time, activated partial thromboplastin time (APTT) was reduced from 94 s before treatment to 78-80 s. Tail bleeding time decreased from 15 min to 1.5-7 min after treatment, some mice being normalised. These findings indicate that the delivered hFIX is biologically active. Similarly treated NOD/SCID mice had circulating hFIX levels of 170 ng/ml on day 1 that remained detectable for 1 month, albeit at low levels. Cell viability of microcapsules retrieved on day 60 was below 5%.
  
  
  CONCLUSIONS
  Our findings indicate that encapsulated human primary myoblasts secrete functional hFIX. Furthermore, implantation of encapsulated human primary myoblasts can partially correct the phenotype of hemophilia B mice, supporting the feasibility of this gene therapy approach for hemophilia B. However, the long-term viability of the encapsulated human myoblasts must first be improved.
authors.affiliation: Research & Development, Canadian Blood Services, McMaster University, Hamilton, Ontario, L8N 3Z5 Canada.
authors.forename: Jianping
authors.initials: J
authors.lastname: Wen
authors.forename: Nong
authors.initials: N
authors.lastname: Xu
authors.forename: Anna
authors.initials: A
authors.lastname: Li
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Bourgeois
authors.forename: Frederick A
authors.initials: FA
authors.lastname: Ofosu
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Hortelano
mesh_terms: D000818:Animals; D002214:Capsules / Q000008:administration & dosage; D002470:Cell Survival; D016503:Drug Delivery Systems / Q000592:standards*; D005164:Factor IX / Q000008:administration & dosage*; D002836:Hemophilia B / Q000628:therapy*; D006801:Humans; D051379:Mice; D032446:Myoblasts / Q000378:metabolism / Q000637:transplantation*; D016896:Treatment Outcome
chemical_list: D002214:Capsules; D005164:Factor IX