Reprogramming Erythroid Cells for Lysosomal Enzyme Production Leads to Visceral and CNS Cross-Correction in Mice with Hurler Syndrome
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Reprogramming erythroid cells for lysosomal enzyme production leads to visceral and CNS cross-correction in mice with Hurler syndrome Daren Wanga, Wei Zhanga, Theodosia A. Kalfab,c, Gregory Grabowskic,d, Stella Daviesc,e, Punam Malika,b,c, and Dao Pana,c,1 aMolecular and Gene Therapy Program, Division of Experimental Hematology and Cancer Biology, bDivision of Hematology/Oncology, dDivision of Human Genetics, and eDivision of Bone Marrow Transplantation and Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; and cDepartment of Pediatrics, University of Cincinnati School of Medicine, Cincinnati, OH 45221 Edited by Inder M. Verma, The Salk Institute for Biological Studies, La Jolla, CA, and approved October 02, 2009 (received for review July 29, 2009) Restricting transgene expression to maturing erythroid cells can However, it is limited by likely poor penetration to the CNS, the reduce the risk for activating oncogenes in hematopoietic stem need for frequent i.v. infusions for a lifetime, and tremendous costs. cells (HSCs) and their progeny, yet take advantage of their robust A therapeutical approach with lower mortality and morbidity, and protein synthesis machinery for high-level protein production. This with the capacity to correct CNS deterioration, is needed. study sought to evaluate the feasibility and efficacy of reprogram- Ex vivo HSC gene transfer followed by autologous transplanta- ming erythroid cells for production of a lysosomal enzyme, ␣-L- tion is an attractive alternative for LSD treatment that could iduronidase (IDUA). An erythroid-specific hybrid promoter pro- provide lifelong therapeutical effects without the morbidity and vided inducible IDUA expression and release during in vitro mortality of allogeneic transplantation. Previously, we showed the erythroid differentiation in murine erythroleukemia cells, resulting feasibility of transducing human primitive hematopoietic progeni- in phenotypical cross-correction in an enzyme-deficient lympho- tors from MPS I patients (6). However, in general, the frequencies blastoid cell line derived from patients with mucopolysaccharidosis of transduced and successfully engrafted HSCs have been low in type I (MPS I). Stable and higher than normal plasma IDUA levels gene therapy clinical trials. Strong in vivo selection pressure for were achieved in vivo in primary and secondary MPS I chimeras for genetically corrected cells appears to be necessary to obtain clini- MEDICAL SCIENCES at least 9 months after transplantation of HSCs transduced with the cally relevant long-term functional corrections, as demonstrated in erythroid-specific IDUA-containing lentiviral vector (LV). More- gene therapy trials for children with inherited immunodeficiencies over, long-term metabolic correction was demonstrated by nor- (7–10). Unfortunately, inadvertent activation of cellular proto malized urinary glycosaminoglycan accumulation in all treated oncogenes by provirus insertion resulted in secondary leukemo- MPS I mice. Complete normalization of tissue pathology was genesis in two otherwise successful clinical trials (11–13). In addi- observed in heart, liver, and spleen. Notably, neurological function tion, selective advantage is not available for most other diseases. and brain pathology were significantly improved in MPS I mice by Restricting transgene expression to late erythroblasts and RBCs erythroid-derived, higher than normal peripheral IDUA protein. may reduce the risk for activating oncogenes in HSCs and their These data demonstrate that late-stage erythroid cells, transduced offspring in all lineages (14, 15). Moreover, healthy individuals can with a tissue-specific LV, can deliver a lysosomal enzyme contin- produce 2.4 ϫ 1011 RBCs/d with a daily output of 7.2 g hemoglobin. uously at supraphysiological levels to the bloodstream and can Redirecting a portion of the formidable protein synthesis machin- correct the disease phenotype in both viscera and CNS of MPS I ery in maturing erythroid cells toward the expression of a transgene mice. This approach provides a paradigm for the utilization of RBC may provide an efficient approach for long-term protein delivery to precursors as a depot for efficient and potentially safer systemic the circulation. The high efficiency of protein synthesis may com- delivery of nonsecreted proteins by ex vivo HSC gene transfer. pensate for the generally low HSC gene transfer frequency. Recent studies by Chang et al. (16) demonstrated the feasibility of long- hematopoietic stem cells ͉ neurological function ͉ gene therapy ͉ term secretion of therapeutical levels of human factor IX in plasma lysosomal storage diseases ͉ RBC precursors from HSC-derived erythroid cells using the human -globin pro- moter/enhancer. Our previous work has identified an ankyrin-1– based erythroid-specific hybrid promoter/enhancer (IHK) that ucopolysaccharidosis type I (MPS I) or Hurler syndrome, a could introduce high erythroid-specific expression in vivo in pri- common lysosomal storage disease (LSD), is caused by M mary and secondary murine BMT recipients (17). defective ␣-L-iduronidase (IDUA) (EC3.2.1.76) and consequent In the present study, we hypothesize that maturing erythroid cells systemic accumulation of the unprocessed glycosaminoglycans derived from genetically modified HSCs can provide long-term (GAGs) (1). Clinical features in patients with MPS I include systemic production of IDUA and lead to phenotypic cross- cardiac, hepatic, and soft tissue defects as well as CNS abnormalities correction in an animal model of MPS I. Using lentiviral vectors in severely affected patients who would die by the age of 10 years (LVs), we demonstrated that the IHK promoter/enhancer provided if untreated. Allogeneic hematopoietic stem cell (HSC) transplan- inducible lysosomal IDUA expression and release during in vitro tation (BMT) from healthy donors provides therapeutical benefits, erythroid differentiation in murine erythroleukemia (MEL) cells, including prolonging life and improving some of the visceral resulting in cross-correction in lymphoblastoid cells derived from an manifestations, by metabolic cross-correction from intercellular enzyme transfer (2). Furthermore, BMT early in life (Ͻ2 years) leads to significant improvement in CNS outcomes, even though Author contributions: G.G., S.D., P.M., and D.P. designed research; D.W., W.Z., T.A.K., and minimal or no response has been obtained for the reversal of D.P. performed research; P.M. contributed new reagents/analytic tools; D.W., W.Z., T.A.K., preexisting CNS abnormalities (2–5). Despite these benefits, allo- and D.P. analyzed data; and D.P. wrote the paper. geneic transplantation is limited by a procedure-related mortality The authors declare no conflict of interest. rate between 20–30%, late complications such as graft-versus-host This article is a PNAS Direct Submission. disease, and the need to find an HLA-matched donor. A pharma- 1To whom correspondence should be addressed. E-mail: [email protected]. ceutical IDUA product is now available and is being used to This article contains supporting information online at www.pnas.org/cgi/content/full/ ameliorate visceral manifestations of MPS I in some patients. 0908528106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908528106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 26, 2021 MPS I patient (LCLmps). We further showed in vivo that stable and higher than normal IDUA activity levels could be achieved in the plasma of primary or secondary MPS I chimeras for at least 9 months after transplantation of MPS I HSCs transduced with an IDUA-containing erythroid-specific LV. Long-term systemic met- abolic correction and complete normalization of visceral pathology were attained in MPS I mice 5 months after treatment. Moreover, significant improvement of neurological function and brain pathol- ogy were achieved in MPS I mice by the erythroid-derived higher than normal peripheral IDUA protein. This approach would pro- vide a paradigm for the utilization of RBC precursors as a depot for efficient and systemic delivery of proteins that are not convention- ally secreted. Results Inducible IDUA Expression and Enzyme Release from IHK Promoter During in Vitro Erythroid Differentiation in MEL Cells. To determine if cells from the erythroid lineage could produce and release lysosomal IDUA during erythroid differentiation, an erythroid MEL cell line was used to compare IDUA expression and enzyme release from 3 LV constructs containing the same expression cassette with 3 different promoters, i.e., erythroid- specific IHK promoter, ubiquitous cellular promoter of human elongation factor (EF)-1␣, and LTR promoter of spleen focus- forming virus (SF) (Fig. 1A). Progressive erythroid differentia- tion during hexamethylene bisacetamide (HMBA) induction of MEL cells was confirmed by morphologic evaluation and by histochemical staining with benzidine showing an increasing number of hemoglobin-expressing cells (Fig. S1). The mean fluorescent intensity (MFI) of GFP in stably transduced MEL- Fig. 1. Transgene expression and release during erythroid induction in MEL KIiG increased from a mean of 27 to 78 by day 8 of inductive cells. (A) Illustration of LVs. PIHK, erythroid-specific hybrid promoter/enhancer culture, whereas the MFI in MEL-EIiG decreased from 228 to containing intron 8 erythroid-specific enhancer of human ALAS2 (I), HS40 core 84 and no significant change of MFI was observed in MEL-SIiG element from human ␣-globin locus control region (H), and human ankyrin-1