Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34+ cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O6-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen.
Andre Larochelle, Uimook Choi, Yan Shou, Nora Naumann, Natalia A. Loktionova, Joshua R. Clevenger, Allen Krouse, Mark Metzger, Robert E. Donahue, Elizabeth Kang, Clinton Stewart, Derek Persons, Harry L. Malech, Cynthia E. Dunbar, Brian P. Sorrentino
Genetic variants of the SLC6A3 gene that encodes the human dopamine transporter (DAT) have been linked to a variety of neuropsychiatric disorders, particularly attention deficit hyperactivity disorder. In addition, the homozygous Slc6a3 knockout mouse displays a hyperactivity phenotype. Here, we analyzed 2 unrelated consanguineous families with infantile parkinsonism-dystonia (IPD) syndrome and identified homozygous missense SLC6A3 mutations (p.L368Q and p.P395L) in both families. Functional studies demonstrated that both mutations were loss-of-function mutations that severely reduced levels of mature (85-kDa) DAT while having a differential effect on the apparent binding affinity of dopamine. Thus, in humans, loss-of-function SLC6A3 mutations that impair DAT-mediated dopamine transport activity are associated with an early-onset complex movement disorder. Identification of the molecular basis of IPD suggests SLC6A3 as a candidate susceptibility gene for other movement disorders associated with parkinsonism and/or dystonic features.
Manju A. Kurian, Juan Zhen, Shu-Yuan Cheng, Yan Li, Santosh R. Mordekar, Philip Jardine, Neil V. Morgan, Esther Meyer, Louise Tee, Shanaz Pasha, Evangeline Wassmer, Simon J.R. Heales, Paul Gissen, Maarten E.A. Reith, Eamonn R. Maher
Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid–derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response.
Gary C. Pien, Etiena Basner-Tschakarjan, Daniel J. Hui, Ashley N. Mentlik, Jonathan D. Finn, Nicole C. Hasbrouck, Shangzhen Zhou, Samuel L. Murphy, Marcela V. Maus, Federico Mingozzi, Jordan S. Orange, Katherine A. High
γ-Retroviral vectors (γRVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between γRV and lentiviral vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of γRVs. By comparing the genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as γRVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by γRVs. This integration-site bias was intrinsic to γRVs, as it was also observed for SIN γRVs that lacked genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate genotoxicity.
Eugenio Montini, Daniela Cesana, Manfred Schmidt, Francesca Sanvito, Cynthia C. Bartholomae, Marco Ranzani, Fabrizio Benedicenti, Lucia Sergi Sergi, Alessandro Ambrosi, Maurilio Ponzoni, Claudio Doglioni, Clelia Di Serio, Christof von Kalle, Luigi Naldini
Elevated plasma concentrations of HDL cholesterol (HDL-C) are associated with protection from atherosclerotic cardiovascular disease. Animal models indicate that decreased expression of endothelial lipase (LIPG) is inversely associated with HDL-C levels, and genome-wide association studies have identified LIPG variants as being associated with HDL-C levels in humans. We hypothesized that loss-of-function mutations in LIPG may result in elevated HDL-C and therefore performed deep resequencing of LIPG exons in cases with elevated HDL-C levels and controls with decreased HDL-C levels. We identified a significant excess of nonsynonymous LIPG variants unique to cases with elevated HDL-C. In vitro lipase activity assays demonstrated that these variants significantly decreased endothelial lipase activity. In addition, a meta-analysis across 5 cohorts demonstrated that the low-frequency Asn396Ser variant is significantly associated with increased HDL-C, while the common Thr111Ile variant is not. Functional analysis confirmed that the Asn396Ser variant has significantly decreased lipase activity both in vitro and in vivo, while the Thr111Ile variant has normal lipase activity. Our results establish that loss-of-function mutations in LIPG lead to increased HDL-C levels and support the idea that inhibition of endothelial lipase may be an effective mechanism to raise HDL-C.
Andrew C. Edmondson, Robert J. Brown, Sekar Kathiresan, L. Adrienne Cupples, Serkalem Demissie, Alisa Knodle Manning, Majken K. Jensen, Eric B. Rimm, Jian Wang, Amrith Rodrigues, Vaneeta Bamba, Sumeet A. Khetarpal, Megan L. Wolfe, Stephanie DerOhannessian, Mingyao Li, Muredach P. Reilly, Jens Aberle, David Evans, Robert A. Hegele, Daniel J. Rader
MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3′ untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a–specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell–based therapies, including cancer immunotherapy.
Eirini P. Papapetrou, Damian Kovalovsky, Laurent Beloeil, Derek Sant’Angelo, Michel Sadelain
Iminoglycinuria (IG) is an autosomal recessive abnormality of renal transport of glycine and the imino acids proline and hydroxyproline, but the specific genetic defect(s) have not been determined. Similarly, although the related disorder hyperglycinuria (HG) without iminoaciduria has been attributed to heterozygosity of a putative defective glycine, proline, and hydroxyproline transporter, confirming the underlying genetic defect(s) has been difficult. Here we applied a candidate gene sequencing approach in 7 families first identified through newborn IG screening programs. Both inheritance and functional studies identified the gene encoding the proton amino acid transporter SLC36A2 (PAT2) as the major gene responsible for IG in these families, and its inheritance was consistent with a classical semidominant pattern in which 2 inherited nonfunctional alleles conferred the IG phenotype, while 1 nonfunctional allele was sufficient to confer the HG phenotype. Mutations in SLC36A2 that retained residual transport activity resulted in the IG phenotype when combined with mutations in the gene encoding the imino acid transporter SLC6A20 (IMINO). Additional mutations were identified in the genes encoding the putative glycine transporter SLC6A18 (XT2) and the neutral amino acid transporter SLC6A19 (B0AT1) in families with either IG or HG, suggesting that mutations in the genes encoding these transporters may also contribute to these phenotypes. In summary, although recognized as apparently simple Mendelian disorders, IG and HG exhibit complex molecular explanations depending on a major gene and accompanying modifier genes.
Stefan Bröer, Charles G. Bailey, Sonja Kowalczuk, Cynthia Ng, Jessica M. Vanslambrouck, Helen Rodgers, Christiane Auray-Blais, Juleen A. Cavanaugh, Angelika Bröer, John E.J. Rasko
Bitter taste–sensing G protein–coupled receptors (type 2 taste receptors [T2Rs]) are expressed in taste receptor cells of the tongue, where they play an important role in limiting ingestion of bitter-tasting, potentially toxic compounds. T2Rs are also expressed in gut-derived enteroendocrine cells, where they have also been hypothesized to play a role in limiting toxin absorption. In this study, we have shown that T2R gene expression in both cultured mouse enteroendocrine cells and mouse intestine is regulated by the cholesterol-sensitive SREBP-2. In addition, T2R stimulation of cholecystokinin (CCK) secretion was enhanced directly by SREBP-2 in cultured cells and in mice fed chow supplemented with lovastatin and ezetimibe (L/E) to decrease dietary sterol absorption and increase nuclear activity of SREBP-2. Low-cholesterol diets are naturally composed of high amounts of plant matter that is likely to contain dietary toxins, and CCK is known to improve dietary absorption of fats, slow gastric emptying, and decrease food intake. Thus, these studies suggest that SREBP-2 activation of bitter signaling receptors in the intestine may sensitize the gut to a low-fat diet and to potential accompanying food-borne toxins that make it past the initial aversive response in the mouth.
Tae-Il Jeon, Bing Zhu, Jarrod L. Larson, Timothy F. Osborne
Hutchinson-Gilford progeria syndrome (HGPS), a rare disease that results in what appears to be premature aging, is caused by the production of a mutant form of prelamin A known as progerin. Progerin retains a farnesyl lipid anchor at its carboxyl terminus, a modification that is thought to be important in disease pathogenesis. Inhibition of protein farnesylation improves the hallmark nuclear shape abnormalities in HGPS cells and ameliorates disease phenotypes in mice harboring a knockin HGPS mutation (LmnaHG/+). The amelioration of disease, however, is incomplete, leading us to hypothesize that nonfarnesylated progerin also might be capable of eliciting disease. To test this hypothesis, we created knockin mice expressing nonfarnesylated progerin (LmnanHG/+). LmnanHG/+ mice developed the same disease phenotypes observed in LmnaHG/+ mice, although the phenotypes were milder, and mouse embryonic fibroblasts (MEFs) derived from these mice contained fewer misshapen nuclei. The steady-state levels of progerin in LmnanHG/+ MEFs and tissues were lower, suggesting a possible explanation for the milder phenotypes. These data support the concept that inhibition of protein farnesylation in progeria could be therapeutically useful but also suggest that this approach may be limited, as progerin elicits disease phenotypes whether or not it is farnesylated.
Shao H. Yang, Douglas A. Andres, H. Peter Spielmann, Stephen G. Young, Loren G. Fong
Previously, several individuals with X-linked SCID (SCID-X1) were treated by gene therapy to restore the missing IL-2 receptor γ (IL2RG) gene to CD34+ BM precursor cells using gammaretroviral vectors. While 9 of 10 patients were successfully treated, 4 of the 9 developed T cell leukemia 31–68 months after gene therapy. In 2 of these cases, blast cells contained activating vector insertions near the LIM domain–only 2 (LMO2) proto-oncogene. Here, we report data on the 2 most recent adverse events, which occurred in patients 7 and 10. In patient 10, blast cells contained an integrated vector near LMO2 and a second integrated vector near the proto-oncogene BMI1. In patient 7, blast cells contained an integrated vector near a third proto-oncogene,CCND2. Additional genetic abnormalities in the patients’ blast cells included chromosomal translocations, gain-of-function mutations activating NOTCH1, and copy number changes, including deletion of tumor suppressor gene CDKN2A, 6q interstitial losses, and SIL-TAL1 rearrangement. These findings functionally specify a genetic network that controls growth in T cell progenitors. Chemotherapy led to sustained remission in 3 of the 4 cases of T cell leukemia, but failed in the fourth. Successful chemotherapy was associated with restoration of polyclonal transduced T cell populations. As a result, the treated patients continued to benefit from therapeutic gene transfer.
Salima Hacein-Bey-Abina, Alexandrine Garrigue, Gary P. Wang, Jean Soulier, Annick Lim, Estelle Morillon, Emmanuelle Clappier, Laure Caccavelli, Eric Delabesse, Kheira Beldjord, Vahid Asnafi, Elizabeth MacIntyre, Liliane Dal Cortivo, Isabelle Radford, Nicole Brousse, François Sigaux, Despina Moshous, Julia Hauer, Arndt Borkhardt, Bernd H. Belohradsky, Uwe Wintergerst, Maria C. Velez, Lily Leiva, Ricardo Sorensen, Nicolas Wulffraat, Stéphane Blanche, Frederic D. Bushman, Alain Fischer, Marina Cavazzana-Calvo