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Selective Gene Expression in Brain Microglia Mediated Via Adeno-Associated Virus Type 2 and Type 5 Vectors

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Selective Gene Expression in Brain Microglia Mediated Via Adeno-Associated Virus Type 2 and Type 5 Vectors Gene Therapy (2003) 10, 657–667 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt RESEARCH ARTICLE Selective gene expression in brain microglia mediated via adeno-associated virus type 2 and type 5 vectors M Cucchiarini1,w, XL Ren1, G Perides2 and EF Terwilliger1 1Division of Experimental Medicine, Harvard Institutes of Medicine and Beth Israel Deaconess Medical Center, Boston, MA, USA; and 2Department of Surgery, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA, USA Microglia represent a crucial cell population in the central examined, an element derived from the gene for the murine nervous system, participating in the regulation and surveil- macrophage marker F4/80 was the most discriminating for lance of physiological processes as well as playing key roles microglia. Gene expression from vectors controlled by this in the etiologies of several major brain disorders. The ability element was highly selective for microglia, both in vitro and in to target gene transfer vehicles selectively to microglia would vivo. To our knowledge, this is the first demonstration of provide a powerful new approach to investigations of selective expression of transferred genes in microglia using mechanisms regulating brain pathologies, as well as enable AAV-derived vectors, as well as the first utilization of the development of novel therapeutic strategies. In this recombinant AAV-5 vectors in any macrophage lineage. study, we evaluate the feasibility of specifically and efficiently These results provide strong encouragement for the applica- targeting microglia relative to other brain cells, using vectors tion of these vectors and this approach for delivering based on two different serotypes of adeno-associated virus therapeutic and other genes selectively to microglia. (AAV) carrying cell-type-specific transcriptional elements to Gene Therapy (2003) 10, 657–667. doi:10.1038/sj.gt.3301925 regulate gene expression. Among a set of promoter choices Keywords: CNS; microglia; gene transfer; rAAV; tissue-specificity Introduction basic brain research, as well as for advancing the goal of gene therapy for CNS disorders. Microglia, the brain’s resident macrophages, are a crucial Attempts to genetically manipulate primary hemato- if minor component of the central nervous system (CNS), poietic cells have principally focused upon transferring representing up to 10% of adult brain cells. In the CNS, genes into pluripotent stem cell populations. Relatively microglia participate in the regulation of diverse physio- little emphasis has been given to targeting more mature logical processes by controlling the development, myeloid lineages, in large part because they are strongly growth, and function of other brain cells. In addition, refractory to most types of gene transfer vehicles, even in microglia constitute a key immune surveillance system vitro. Similarly, gene transfer into brain cell populations responsible for destroying infectious agents, removing has focused largely upon the neurons. Nevertheless, cell debris, and promoting brain tissue repair. As a some meaningful if tentative steps in the direction of consequence of their surveillance functions, microglia engineering microglia have been taken. Implantation of become activated in response to many different patho- ex vivo engineered microglial cell lines, or precursor cells, logic processes in the CNS, including AIDS dementia, has been successfully performed in several animal Alzheimer’s disease, multiple sclerosis, ischemia, and models.1,2 In another novel approach, engraftment into Creutzfeldt–Jakob disease. In turn, inappropriate or mouse brain of a neural stem cell line engineered to shed chronic over-activation of microglia is believed to play a retroviral vector resulted in gene transfer into the a key role in advancing the pathologies of several of microglia compartment.3 these diseases. In addition, microglia represent the major To approach this task, we elected to use recombinant cell type in the brain susceptible to infection by HIV. vectors based upon adeno-associated virus (rAAV) as Owing to their central roles in controlling brain injury gene transfer vehicles. AAV represents a small family of and disease, and their susceptibility to subversion in a replication-defective human parvoviruses not associated set of diverse, clinically important pathogenic processes, with any pathologies. Typical AAV vectors retain none of microglia constitute a pivotal population for study in the viral coding sequences,4 are nontoxic, and exhibit low immunogenicity,5,6 in marked contrast with other classes Correspondence: Dr EF Terwilliger, Division of Experimental Medicine, of viral vectors (adenoviruses, herpes simplex virus).7–9 Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115, The packaging limits of AAV-derived vectors are USA w comparatively small (about 5.5 kb), making inclusion of Current address: Laboratory of Experimental Orthopaedics, Department of Orthopaedic Surgery, Saarland University Medical Center, 66421 very long genomic DNA sequences impractical, but this Homburg, Germany is still fully adequate for many applications. rAAV can be Received 14 January 2002; accepted 13 September 2002 employed to transfer genes into cells of human origin as Targeting transgene expression to microglia with AAV M Cucchiarini et al 658 well as those of other primates and rodents. In human elements derived from genes encoding several different cells, the wild-type AAV genome integrates specifically myeloid-specific antigens: human CD11b and CD68, as within a short region of chromosome 19. However, well as murine F4/80. The properties of these vectors vectors deleted for the viral coding sequences integrate were compared, and contrasted against control vectors slowly and nonspecifically by a different mechanism.10 driven by the high level, nondiscriminatory CMV-IE Therefore, rAAV-delivered transgenes persist in target promoter. Transgene expression was monitored in each cells as a mixture of stable episomes and genuine case using two distinct and complementary indicator integrants, the relative proportions of which vary genes, lacZ and red fluorescent protein (RFP). Expression depending upon the cell type. However, unlike retro- from each vector was evaluated in vitro in microglia vis-a`- viruses, the episomal forms are also actively tran- vis other brain cells, as well as in a different macrophage scribed.11 lineage. While the efficiency of rAAV-mediated gene transfer Among the regulatory elements evaluated, the element varies widely between different cell types, this class of derived from the F4/80 sequence yielded the highest vector has been used successfully to transduce several level of specificity for microglia in primary cultures in primary cell types and tissues which are refractory to vitro, and provided a similar level of selectivity in the most viral vectors, including striated muscle, liver intact rat brain. Combined with the other unique hepatocytes, and vessel endothelium.5,6,12,13 Of particular properties of AAV, these results provide a strong relevance for this study, rAAV have been employed with foundation for a workable approach to effecting micro- good success by many groups to deliver genes into glia-specific gene transfer in living animals. neuron populations in vitro and in cultured brain sections,14 as well as in the intact mammalian CNS.15–18 Also, while there has been little exploration of the utility Results of rAAV to effect gene transfer into differentiated macrophage populations, such as microglia, we pre- Vector design and construction viously employed an rAAV successfully on mature To attempt to restrict the expression of rAAV transgenes alveolar macrophages, as well as peripheral blood to microglia, several candidate transcription elements monocytes.12,19 were selected for the study. The tissue specificities and Most rAAV generated to date have been derived from expression patterns of the human CD11b and CD68 serotype 2 of the virus. In vivo, transduction with rAAV-2 antigens, as well as the murine F4/80, have been vectors in the brain tends to occur primarily into the analyzed in different types of myeloid cells, and each neurons,15,20 in contrast to the in vitro situation, where has been identified as a specific marker of particular astrocytes and oligodendrocytes are also readily trans- myeloid lineages including, to varying degrees, micro- duced.21,22 However, successful targeting of an rAAV-2 to glia.25–28 The regulatory as well as coding sequences of oligodendrocytes in intact brain has also been reported, each gene have also been cloned.28–30 using the vector in concert with a strongly cell-type- The first candidate, CD11b, the a subunit of the specific promoter.22 CD11b/CD18 integrin adhesion molecule, is not present More recently, as additional serotypes of AAV have on the surfaces of immature myeloid precursors, but been cloned and characterized, vectors derived from expression increases during early cell differentiation and several other types of AAV have begun to be exploited. is subsequently restricted to mature monocytes, macro- Sequence alignments and other analyses indicate that phages, neutrophils, natural killer cells, and micro- AAV-5 is the most divergent of the known serotypes of glia.26,29 CD11b is also expressed at low levels in a few AAV.23,24 Of particular note, rAAV-5 vectors appear able other cell types, including fetal liver stem cells, acti- to deliver genes not only into neurons,
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