1 Delivery of nucleic acid therapeutics by genetically engineered hematopoietic 2 stem cells��� 3 Christopher B. Doering, David Archer, H. Trent Spencer ��� 4 Aflac Cancer Center and Blood Disorders Service, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA Q1 5 6 a b s t r a c t a r t i c l e i n f o 7 Article history: 8 Received 14 June 2010 9 Accepted 8 September 2010 10 Available online xxxx 11 12 13 14 Keywords: 15 Hematopoietic stem cell 16 Gene therapy 17 Recombinant lentivirus 18 Hemophilia A 19 Induced pluripotent stem cell 20 Several populations of adult human stem cells have been identified, but only a few of these are in routine 21 clinical use. The hematopoietic stem cell (HSC) is arguably the most well characterized and the most routinely 22 transplanted adult stem cell. Although details regarding several aspects of this cell's phenotype are not well 23 understood, transplant of HSCs has advanced to become the standard of care for the treatment of a range of 24 monogenic diseases and several types of cancer. It has also proven to be an excellent target for genetic 25 manipulation, and clinical trials have already demonstrated the usefulness of targeting this cell as a means of 26 delivering nucleic acid therapeutics for the treatment of several previously incurable diseases. It is anticipated 27 that additional clinical trials will soon follow, such as genetically engineering HSCs with vectors to treat 28 monogenic diseases such as hemophilia A. In addition to the direct targeting of HSCs, induced pluripotent 29 stem (iPS) cells have the potential to replace virtually any engineered stem cell therapeutic, including HSCs. 30 We now know that for the broad use of genetically modified HSCs for the treatment of non-lethal diseases, e.g. 31 hemophilia A, we must be able to regulate the introduction of nucleic acid sequences into these target cells. 32 We can begin to refine transduction protocols to provide safer approaches to genetically manipulate HSCs and 33 strategies are being developed to improve the overall safety of gene transfer. This review focuses on recent 34 advances in the systemic delivery of nucleic acid therapeutics using genetically modified stem cells, 35 specifically focusing on i) the use of retroviral vectors to genetically modify HSCs, ii) the expression of fVIII 36 from hematopoietic stem cells for the treatment of hemophilia A, and iii) the use of genetically engineered 37 hematopoietic cells generated from iPS cells as treatment for disorders of hematopoiesis. 38 �� 2010 Elsevier B.V. All rights reserved. 3940 41 42 43 44 Contents 45 1. Hematopoietic stem cell gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 46 2. Safety issues relating to the genetic engineering of HSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 47 3. Clinical trials utilizing genetically engineered HSCs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 48 4. HSC transplant (HSCT) gene therapy of hemophilia A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 49 5. Alternate sources of HSCs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 50 6. Disease candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 51 7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 52 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 53 54 1. Hematopoietic stem cell gene therapy 55 Hematopoietic stem cells (HSCs) have been a target of genetic 56 engineering from the earliest gene transfer studies, and they continue to 57 be excellent candidates for systemic delivery of many nucleic acid-based 58 therapeutics. The reason for the continued interest is that these cells have 59 the ability to regenerate the entire hematopoietic system, which includes 60 all lineages of blood cells such as lymphocytes and monocytes. The HSC is 61 also readily manipulated ex vivo, which allows for rapid testing of various Advanced Drug Delivery Reviews xxx (2010) xxx���xxx ��� This review is part of the Advanced Drug Delivery Reviews theme issue on ���Stem Cell Gene Manipulation and Delivery as Systemic Therapeutics���. ��� Corresponding author. 2015 Uppergate Drive, Aflac Cancer Center and Blood Disorders Service, Emory Children's Center, Room 420, Emory University, Atlanta, GA, USA. E-mail address: hspence@emory.edu (H.T. Spencer). ADR-12048 No of Pages 9 0169-409X/$ ��� see front matter �� 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2010.09.005 Contents lists available at ScienceDirect Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr Please cite this article as: C.B. Doering, et al., Delivery of nucleic acid therapeutics by genetically engineered hematopoietic stem cells, Adv. Drug Deliv. Rev. (2010), doi:10.1016/j.addr.2010.09.005

62 parameters relating to gene transfer and the expression of the transferred 63 nucleic acid, i.e. the transgene. Importantly, the stable introduction of a 64 transgene into the HSC can result in the expression of the gene product in 65 the progenitor cells derived from the HSC as well as every blood cell 66 derived from the progenitor cells, which can amplify the therapeutic 67 potential of genetically modified HSCs a thousand fold. Many platforms 68 are now available for introducing nucleic acid sequences into HSCs, some 69 of which are currently in clinical trials [1���7]. 70 Successful initial gene transfer preclinical studies used ��-retroviral 71 vectors, such as the Moloney murine leukemia virus (MLV), as nucleic 72 acid-transfer vehicles. The goal of early studies was to develop methods 73 that resulted in 1) stable integration of the transgene into the 74 chromosome of the target cell, 2) efficient nucleic acid transfer, 75 3) engraftment of significant numbers of genetically modified HSC, and 76 4) high-level transgene expression. The mouse proved to be an excellent 77 model system for testing novel gene transfer methods, and ��-retroviral 78 vectorsquickly becamethe gene transfertool ofchoice. Thisrecombinant 79 ��-retroviral vector system is composed of an expression vector, which 80 contains the transgene of interest, and a packaging cell, which is typically 81 based on cell lines such as NIH3T3 or HEK293T cells. Packaging cells are 82 engineered to express the protein components of the ��-retroviruses, 83 which produces a recombinant viral vector capable of transferring 84 virtually any transgene of interest [1,5,7]. 85 Initial studies targeting mammalian HSCs showed inefficient levels 86 of gene transfer. It was quickly shown that understanding the biology 87 of the target cells was critical to accomplishing efficient gene transfer. 88 HSCs are mainly quiescent and divide relatively infrequently. 89 However, ��-retroviruses require cellular division for successful 90 integration of the transgene into the genome of the HSC. Therefore, 91 various protocols were developed that induced HSC division, most of 92 which focused on identifying effective cytokine cocktails that resulted 93 in efficient murine HSC transduction (N50% transduction of murine 94 HSCs are now readily attained [8���12]). In addition, the introduction of 95 fibronectin fragments was included to the transduction protocol, 96 which further enhanced the efficiency of gene transfer, presumably 97 through co-localization of viral vectors and target cells thereby 98 increasing the effective concentrations of each [13���15]. 99 2. Safety issues relating to the genetic engineering of HSCs 100 With efficient packaging cells in hand generating high titers of 101 recombinant virus combined with efficient transduction protocols, HSC 102 genetic engineering quickly moved from preclinical studies to clinical 103 trials. The initial gene therapy trials focused on immunodeficiency 104 disorders, and a clinical trial designed to treat childhood severe combined 105 immune deficiency (SCID) X1 disease provided direction for the entire 106 genetherapy field.Inthistrial,aseriousadverseeventwasreportedinlate 107 2002, which described the development of leukemia in one of the treated 108 patients [16]. Although 9 of 10 children enrolled in the trial showed 109 correction of the immune deficiency for more than five years, an initial 110 patient developed a T-cell leukemia-like illness three years after receiving 111 genetically modified HSCs. Additional children subsequently developed a 112 similar disease, and the FDA put a clinical hold on similar trials. Of the 20 113 treated children, 5 have developed the T-cell leukemia-like disorder. The 114 cause of the leukemia is now well documented, and several outstanding 115 studies have described the molecular events resulting in the leukemia 116 [16,17]. Briefly, the transferred nucleic acid sequence in the SCID X1 trials 117 encoded the common �� chain of the cytokine receptor subunit, which is a 118 component of the IL-2, IL-4, IL-7, IL-9, IL-11, IL-15 and IL-21 signaling 119 receptors. The cDNA was transferred using a recombinant �� retroviral 120 vector. It is now known that ��-retroviruses integrate into chromosomal 121 sites of active transcription [18���22]. For the children who developed 122 leukemia, the transferred gene integrated near known proto-oncogenes, 123 such as LMO-2. These insertional mutagenic events caused aberrant 124 transcription and expression of the oncogene, and when combined with 125 the enforced expression of the �� chain of the cytokine receptor led to the 126 dysregulation of T-cell expansion and leukemia. Prior to this study, it was 127 predicted that retroviruses integrated randomly. However, it is now well 128 documented that retroviruses can cause severe adverse events through 129 mechanisms associated with insertional mutagenesis. Therefore, investi- 130 gations focusing on insertion-site analysis have been a major priority in 131 the field of HSC-directed gene therapy. From these studies, it is now 132 clear that under certain circumstances recombinant viruses can alter 133 endogenous gene function at the vicinity of viral nucleic acid integration. 134 A major issue, therefore, facing the field of HSC-directed gene 135 therapy is that of insertional mutagenesis (Fig. 1). Some of these 136 concerns have been alleviated with the introduction of safety- 137 engineered gene transfer vectors. Recombinant lentiviruses, such as 138 those derived from the human immunodeficiency virus (HIV), are now 139 known to be less genotoxic compared to the �� retroviral vectors used in 140 the initial gene transfer studies. In support of using lentivirus-based 141 vectors, during the past 5 years, VIRxSYS Corp. (Gaithersburg, MD) has 142 transduced and transplanted over a trillion HIV-1-based recombinant 143 lentivirus-transduced CD4+ cells in their phase 1 clinical trials for Fig. 1. Schematic showing the integration of three viral vectors into the genomic DNA of a target cell. The first vector, a MLV-based vector, integrates near the promoter of gene 1. Potential results of integrating near or within promoter sequences are the dysregulation of the downstream gene. In addition, the enhancer activity of the MLV promoter can activate nearby genes (within ~300 kb), such as gene 2 in the figure. The second vector, an HIV-based vector, also integrates into gene 1, but instead of targeting the promoter region, recombinant HIV vectors integrate within the gene, typically downstream of the promoter. This integration pattern can disrupt expression of the gene in which the vector integrates, e.g. gene 1, and similar to the MLV vector nearby genes can also be disrupted, such as gene 2 within the figure. The third vector, an HIV-SIN-based vector, integrates within a gene sequence, similar to HIV vectors, and will potentially disrupt expression of the targeted gene, which is gene 3 in the figure. Because the viral LTR promoter sequences are inactivated expression of nearby genes, such as gene 4 in the figure, are not affected. 2 C.B. Doering et al. / Advanced Drug Delivery Reviews xxx (2010) xxx���xxx Please cite this article as: C.B. Doering, et al., Delivery of nucleic acid therapeutics by genetically engineered hematopoietic stem cells, Adv. Drug Deliv. Rev. (2010), doi:10.1016/j.addr.2010.09.005