High-level transgene expression in induced pluripotent stem cell–derived megakaryocytes: correction of Glanzmann thrombasthenia

Abstract

• When targeted to a single allele of the AAVS1 locus, the Gp1ba promoter drives a high level of expression specifically to megakaryocytes.• Transgene rescue in iPSCs provides a model for the return of surface aIIbb3 expression to near-normal levels in patients with type I GT.Megakaryocyte-specific transgene expression in patient-derived induced pluripotent stem cells (iPSCs) offers a new approach to study and potentially treat disorders affecting megakaryocytes and platelets. By using a Gp1ba promoter, we developed a strategy for achieving a high level of protein expression in human megakaryocytes. The feasibility of this approach was demonstrated in iPSCs derived from two patients with Glanzmann thrombasthenia (GT), an inherited platelet disorder caused by mutations in integrin aIIbb3. Hemizygous insertion of Gp1ba promoter-driven human aIIb complementary DNA into the AAVS1 locus of iPSCs led to high aIIb messenger RNA and protein expression and correction of surface aIIbb3 in megakaryocytes. Agonist stimulation of these cells displayed recovery of integrin aIIbb3 activation. Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function. (Blood. 2014;123(5):753-757)

Comparative analysis of human ex vivo–generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale

“…An iPSC line (WTBM1-8) was generated using a lentivirus expressing OCT4, SOX2, KLF4, and MYC 22 and were analyzed for pluripotency by teratoma formation, flow cytometry, and gene expression. 23 iPSCs were differentiated into megakaryocytes as previously described. 24 For all, large megakaryocytes were isolated using a 2-step bovine serum albumin density gradient described for murine megakaryocytes 19 and counted by hematocytometer before retro-orbital infusion in 200 mL phosphate-buffered saline (PBS; Invitrogen).…”

Megakaryocyte- and megakaryocyte precursor–related gene therapies

“…31,32,45,72 This work has furthered our efforts to achieve the ultimate goal of correcting inherited genetic defects of platelets and permitting platelets to deliver therapeutic agents directly to the site of injury. There remains an endless challenge to increase the safety and benefit from HSC gene transfer that will likely concentrate on developing strategies that obtain high transduction and gene marking efficiencies with low frequency for insertional mutagenesis.…”

“…32 Similar to HSC gene transfer and transplant, this approach resulted in the de novo expression of 50% of normal levels of a functional aIIbb3 receptor on the surface of megakaryocytes in vitro. 32 The results from this work indicate that transfusion of gene-modified autologous platelets derived from iPSCs could provide additional elements of safety for a gene transfer protocol because the potential for the patient to develop mutagenesis and oncogenesis due to pretransplant conditioning reagents and random insertion of a gene transfer vector into the genome is greatly reduced by transfusion of anucleate platelets. In addition, transplant of autologous platelets with 1 new receptor may decrease immunemediated recognition and destruction of transfused platelets as observed with transfusions from unrelated donors.…”

“…Thus, recent studies describing transfusion of platelets derived from iPSCs or HSCs modified with gene transfer vectors indicate the potential feasibility toward accomplishing this goal. 32,77,78 Current analysis of the field indicates that it is essential to achieve further improvement for in vitro production of platelets (especially to increase the number and quality of platelets for transfusion) to make this strategy clinically feasible. 79 The authors of that article surmised that it may be more advantageous to infuse megakaryocytes (rather than platelets) derived from iPSCs.…”

“…To date, the most common megakaryocyte-specific gene promoters used within gene transfer vectors used for megakaryocyte modification include: GP1BA, ITGA2B, and PF4 because they have been shown to consistently drive moderate-to high-level protein expression preferentially within megakaryocytes (see Table 1). [31][32][33] These gene promoters could potentially be used to drive megakaryocyte-specific transgene expression within the confines of a variety of gene transfer vectors that have been characterized for their ability to efficiently and safely express the transgene product including: recombinant lentiviral, adenoviral, and adeno-associated viral vectors as well as plasmid DNA. 34 Each gene transfer technique has unique advantages and disadvantages depending partially upon vector used and the disorder that is being treated (summarized in Table 1).…”