commentary
© The American Society of Gene & Cell Therapy
See page 617
A Shot in the Bone Corrects a Genetic Disease Brian D Brown1 doi:10.1038/mt.2015.38
T
remendous success has now been achieved for the treatment of adenosine deaminase deficiency severe combined immunodeficiency,1 metachromatic leukodystrophy,2 and other severe monogenic diseases through the use of ex vivo hematopoietic stem and progenitor cell gene transfer (HSPC-GT) and subsequent bone marrow transplant (BMT)3 of the genecorrected cells. In HSPC-GT/BMT, enriched preparations of hematopoietic stem cells (HSPCs) are collected from patients and transduced ex vivo with a lentiviral vector (LV) or retroviral vector encoding the defective gene. The cells are then transplanted back into patients that have been preconditioned with myeloablative agents so as to deplete the recipient’s existing hematopoietic cells, thereby making “space” for the transduced stem cells to engraft and repopulate the patient’s hematopoietic system with cells expressing a functional copy of the defective gene. The promise of HSPC-GT/BMT has led to an interest in using this approach for the treatment of hemophilia A and B.4–6 One of the most exciting developments in the use of HSPC-GT/BMT for hemophilia was work by Montgomery and colleagues that demonstrated that transduction of HSPCs with an LV expressing factor VIII (FVIII) under the control of the glycoprotein αIIb (GPαIIb) promoter restricted FVIII expression to megakaryocytes following transplant.7,8 This had the important benefit that FVIII was enriched in megakaryocyte-derived platelets, which were able to release the clotting factor locally into wounds, so 1 Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York USA. Correspondence: Brian Brown, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 1470 Madison Avenue, New York, New York 10029, USA. E-mail: [email protected]
614
as to circumvent neutralization by preexisting FVIII antibodies. However, despite the efficacy of HSPC-GT/BMT in preclinical models of hemophilia, there has yet to be a clinical trial of the approach. Because a factor VIII– or factor IX–encoding LV is expected to have a low oncogenic potential, the possibility of insertional mutagenesis may not be a major hurdle for the translation of this approach. A more definite concern is over the need for myeloablative conditioning in hemophiliacs. BMT carries significant risks, including a high susceptibility to infections and even death. Unlike the case with an untreatable disease such as metachromatic leukodystrophy, for patients with hemophilia these risks are cause for trepidation. In this issue of Molecular Therapy, Wang and colleagues9 describe a clever approach for hemophilia HSPC-GT that circumvents the need for ablative conditioning. Specifically, they show that direct injection of LVs into the bone marrow (intraosseous, IO) of the tibia of mice can stably transfer a FVIII transgene into HSPCs and their progeny without the need for BMT. Initially they evaluated the efficiency of IO delivery using a LV encoding green fluorescent protein from the ubiquitously expressed human elongation factor 1α promoter, and found that more than 15% of HSPCs (defined as Lin−Sca1+c-Kit+ cells) were transduced. They then inserted a FVIII transgene into the LV and delivered it IO into hemophilia A mice. They were able to detect FVIII production in up to 2% of HSPCs and even to measure FVIII activity in circulating blood. However, FVIII expression was lost over time, and antibodies to FVIII developed in the mice. To overcome this problem, Wang et al. adopted a platelet-targeted expression approach.7,8 They substituted the broadly active human elongation factor 1α promoter with
the glycoprotein 1bα (Gp1bα) promoter, whose activity is limited to late-stage megakaryocytes, and injected the vector IO. Although this did not affect the vector’s pattern of transduction, FVIII was no longer expressed in HSPCs or other differentiated cells, but was detected in ~2% of CD42d+ platelets. These findings are consistent with the pattern of expression of the Gp1bα promoter. Importantly, these mice exhibited reduced blood loss upon tail clipping. Even more impressively, when the Gp1bα-driven LV was injected IO into hemophilia A mice that had preexisting FVIII antibodies, blood clotting times were still reduced. This indicated that the vector and this delivery approach could mediate functional correction of the bleeding phenotype even in the presence of FVIII inhibitors. Before IO delivery of LVs can be moved to the clinic, several issues will need to be addressed. In particular, it will be important to determine whether IO injection of the vector causes any disruptions to hematopoiesis. LVs can trigger a transient type I interferon response through activation of plasmacytoid dendritic cells, which are relatively abundant in bone marrow.10 This could be dangerous to patients, though it may be manageable with anti-inflammatory agents.10,11 A long-term concern is that transduction of cells in the stem cell niche could somehow affect the biology of the HSPCs, or that this approach could increase the risk of insertional mutagenesis. It must also be determined whether long-term HSPCs are actually being transduced by IO injection. These issues will require more extended follow-up and even serial transplantation studies. Nonetheless, the work by Wang et al. represents a promising new approach for hemophilia A gene therapy that overcomes several hurdles inherent in existing approaches—in particular, the need for myeloablative conditioning. This strategy could one day benefit patients with hemophilia A and other genetic diseases that have thus far not been considered for HSPC gene transfer. Furthermore, this novel in vivo approach could be considered as an alternative therapy for some of the diseases currently treated by LV gene transfer to HSPCs using conventional ex vivo protocols. www.moleculartherapy.org vol. 23 no. 4 april 2015
© The American Society of Gene & Cell Therapy
references
1. Aiuti, A, Cattaneo, F, Galimberti, S, Benninghoff, U, Cassani, B, Callegaro, L et al. (2009). Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N Engl J Med 360: 447–458. 2. Biffi, A, Montini, E, Lorioli, L, Cesani, M, Fumagalli, F, Plati, T et al. (2013). Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science 341: 1233158. 3. Naldini, L (2011). Ex vivo gene transfer and correction for cell-based therapies. Nat Rev Genet 12: 301–315. 4. Chuah, MK, Evens, H and VandenDriessche, T (2013). Gene therapy for hemophilia. J Thromb Haemost 11 (suppl. 1): 99–110. 5. Moayeri, M, Ramezani, A, Morgan, RA, Hawley, TS and Hawley, RG (2004). Sustained phenotypic correc-
commentary
tion of hemophilia A mice following oncoretroviralmediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells. Mol Ther 10: 892–902. 6. Follenzi, A, Raut, S, Merlin, S, Sarkar, R and Gupta, S (2012). Role of bone marrow transplantation for correcting hemophilia A in mice. Blood 119: 5532–5542. 7. Shi, Q, Wilcox, DA, Fahs, SA, Fang, J, Johnson, BD, Du, LM et al. (2007). Lentivirus-mediated platelet-derived factor VIII gene therapy in murine haemophilia A. J Thromb Haemost 5: 352–361. 8. Kuether, EL, Schroeder, JA, Fahs, SA, Cooley, BC, Chen, Y, Montgomery, RR et al. (2012). Lentivirusmediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity. J Thromb Haemost 10: 1570–1580.
9. Wang, X, Shin, SC, Chiang, AFJ, Khan, I, Pan, D, Rawlings, DJ et al. (2015). Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A. Mol Ther 23: 617–626 10. Agudo, J, Ruzo, A, Kitur, K, Sachidanandam, R, Blander, JM and Brown, BD (2012). A TLR and non-TLR mediated innate response to lentiviruses restricts hepatocyte entry and can be ameliorated by pharmacological blockade. Mol Ther 20: 2257–2267. 11. Wang, CX, Sather, BD, Wang, X, Adair, J, Khan, I, Singh, S et al. (2013). Rapamycin relieves lentiviral vector transduction resistance in human and mouse hematopoietic stem cells. Blood 124: 913–923.
See page 707
Antiobesity Strategy Targets Energy Economy Safeguards Michel Vivaudou1–3 and André Terzic4 doi:10.1038/mt.2015.39
A
fundamental barrier in obesity management is that caloric restriction triggers energy-conserving responses that evolved to prevent body weight loss. ATPsensitive potassium (KATP) channels have been identified as safeguards controlling energy expenditure in skeletal muscles and thereby key factors determining body weight.1 In this issue of Molecular Therapy, Koganti and colleagues report the successful reduction of muscle energy efficiency through targeted intramuscular injections of cell-penetrating vivo-morpholinos to prevent translation of the channel poreforming Kir6.2 subunit.2 In this elegant proof-of-concept study, the authors demonstrate localized reduction of KATP channel expression and function, leading in turn to an increase in activity-related energy consumption, without compromising exercise tolerance. This report opens a new avenue of investigation in targeted 1 Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France; 2Centre National de la Recherche Scientifique, Institut de Biologie Structurale, Grenoble, France; 3CEA, Institut de Biologie Structurale, Grenoble, France; 4Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA Correspondence: Michel Vivaudou, Institut de Biologie Structurale, 71 Avenue des Martyrs, 38044 Grenoble, France. E-mail: vivaudou@ ibs.fr or André Terzic, Center for Regenerative Medicine, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA. E-mail: [email protected]
Molecular Therapy vol. 23 no. 4 april 2015
therapies aiming to control weight management. Obesity reflects an imbalance between calorie intake and expenditure. At present, more than 1 billion adults worldwide are considered overweight, underscoring a rampant epidemic.3 The global prevalence of obesity has precipitated a major escalation in comorbidities associated with an increase in overall mortality.4 Beyond intensive counseling and change in lifestyle, diverse strategies targeting weight loss are being pursued.5 Progress is reflected by US Food and Drug Administration approval of new medications for chronic weight management in obese patients, such as lorcaserin and phentermine/topiramate. Yet antiobesity drug therapy has been largely unsuccessful because of lack of efficacy, poor adherence, and adverse effects.6 Elucidating the molecular pathways that underlie the caloric intake–expenditure equilibrium is necessary to inform the selection of promising therapeutic targets. Indeed, a deeper insight into the innate mechanisms regulating appetite, nutrient exposure, and energy balance is warranted to aid in the discovery and future development of next-generation therapies. Stimulation of energy expenditure may be a potent strategy for obesity treatment. KATP channels, expressed at high density in striated muscles and other excitable tissues, are established membrane sensors of ATP/ADP.7–9 It has been postulated that
under conditions of energy deficit, activation of channel complexes would result in protective energy economy, whereas under energy surplus, downregulation of KATP channels would increase thermogenesis.10 As such, KATP channels provide a low-fuel warning that signals muscle fibers to slow down and avoid irreversible energy depletion. Through tight regulation by adenine nucleotides and integration with metabolic pathways, muscle KATP channels seem to sense both static metabolic levels and the dynamics of energy consumption, thus maintaining an optimal balance between lost heat production and useful mechanical work.11 Without functional KATP channels, energy efficiency decreases and muscles burn more calories than normal.1 Thus, weight loss could be achieved without additional exercise, by “simply” reducing the activity of skeletal muscle KATP channels. A pharmacological approach is conceivable, as numerous molecules are known to modulate KATP channels by binding to their regulatory SUR subunit.12 Unfortunately, blockers of KATP channels specific for skeletal muscle do not yet exist and are unlikely to be discovered soon, given that comparable channels (incorporating isoform SUR2) are also found in cardiac and smooth muscle.13 In the new work, Koganti and colleagues2 used an alternative approach of reducing protein levels by leveraging antisense oligonucleotides packaged as a 615
© The American Society of Gene & Cell Therapy
See page 617
A Shot in the Bone Corrects a Genetic Disease Brian D Brown1 doi:10.1038/mt.2015.38
T
remendous success has now been achieved for the treatment of adenosine deaminase deficiency severe combined immunodeficiency,1 metachromatic leukodystrophy,2 and other severe monogenic diseases through the use of ex vivo hematopoietic stem and progenitor cell gene transfer (HSPC-GT) and subsequent bone marrow transplant (BMT)3 of the genecorrected cells. In HSPC-GT/BMT, enriched preparations of hematopoietic stem cells (HSPCs) are collected from patients and transduced ex vivo with a lentiviral vector (LV) or retroviral vector encoding the defective gene. The cells are then transplanted back into patients that have been preconditioned with myeloablative agents so as to deplete the recipient’s existing hematopoietic cells, thereby making “space” for the transduced stem cells to engraft and repopulate the patient’s hematopoietic system with cells expressing a functional copy of the defective gene. The promise of HSPC-GT/BMT has led to an interest in using this approach for the treatment of hemophilia A and B.4–6 One of the most exciting developments in the use of HSPC-GT/BMT for hemophilia was work by Montgomery and colleagues that demonstrated that transduction of HSPCs with an LV expressing factor VIII (FVIII) under the control of the glycoprotein αIIb (GPαIIb) promoter restricted FVIII expression to megakaryocytes following transplant.7,8 This had the important benefit that FVIII was enriched in megakaryocyte-derived platelets, which were able to release the clotting factor locally into wounds, so 1 Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York USA. Correspondence: Brian Brown, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 1470 Madison Avenue, New York, New York 10029, USA. E-mail: [email protected]
614
as to circumvent neutralization by preexisting FVIII antibodies. However, despite the efficacy of HSPC-GT/BMT in preclinical models of hemophilia, there has yet to be a clinical trial of the approach. Because a factor VIII– or factor IX–encoding LV is expected to have a low oncogenic potential, the possibility of insertional mutagenesis may not be a major hurdle for the translation of this approach. A more definite concern is over the need for myeloablative conditioning in hemophiliacs. BMT carries significant risks, including a high susceptibility to infections and even death. Unlike the case with an untreatable disease such as metachromatic leukodystrophy, for patients with hemophilia these risks are cause for trepidation. In this issue of Molecular Therapy, Wang and colleagues9 describe a clever approach for hemophilia HSPC-GT that circumvents the need for ablative conditioning. Specifically, they show that direct injection of LVs into the bone marrow (intraosseous, IO) of the tibia of mice can stably transfer a FVIII transgene into HSPCs and their progeny without the need for BMT. Initially they evaluated the efficiency of IO delivery using a LV encoding green fluorescent protein from the ubiquitously expressed human elongation factor 1α promoter, and found that more than 15% of HSPCs (defined as Lin−Sca1+c-Kit+ cells) were transduced. They then inserted a FVIII transgene into the LV and delivered it IO into hemophilia A mice. They were able to detect FVIII production in up to 2% of HSPCs and even to measure FVIII activity in circulating blood. However, FVIII expression was lost over time, and antibodies to FVIII developed in the mice. To overcome this problem, Wang et al. adopted a platelet-targeted expression approach.7,8 They substituted the broadly active human elongation factor 1α promoter with
the glycoprotein 1bα (Gp1bα) promoter, whose activity is limited to late-stage megakaryocytes, and injected the vector IO. Although this did not affect the vector’s pattern of transduction, FVIII was no longer expressed in HSPCs or other differentiated cells, but was detected in ~2% of CD42d+ platelets. These findings are consistent with the pattern of expression of the Gp1bα promoter. Importantly, these mice exhibited reduced blood loss upon tail clipping. Even more impressively, when the Gp1bα-driven LV was injected IO into hemophilia A mice that had preexisting FVIII antibodies, blood clotting times were still reduced. This indicated that the vector and this delivery approach could mediate functional correction of the bleeding phenotype even in the presence of FVIII inhibitors. Before IO delivery of LVs can be moved to the clinic, several issues will need to be addressed. In particular, it will be important to determine whether IO injection of the vector causes any disruptions to hematopoiesis. LVs can trigger a transient type I interferon response through activation of plasmacytoid dendritic cells, which are relatively abundant in bone marrow.10 This could be dangerous to patients, though it may be manageable with anti-inflammatory agents.10,11 A long-term concern is that transduction of cells in the stem cell niche could somehow affect the biology of the HSPCs, or that this approach could increase the risk of insertional mutagenesis. It must also be determined whether long-term HSPCs are actually being transduced by IO injection. These issues will require more extended follow-up and even serial transplantation studies. Nonetheless, the work by Wang et al. represents a promising new approach for hemophilia A gene therapy that overcomes several hurdles inherent in existing approaches—in particular, the need for myeloablative conditioning. This strategy could one day benefit patients with hemophilia A and other genetic diseases that have thus far not been considered for HSPC gene transfer. Furthermore, this novel in vivo approach could be considered as an alternative therapy for some of the diseases currently treated by LV gene transfer to HSPCs using conventional ex vivo protocols. www.moleculartherapy.org vol. 23 no. 4 april 2015
© The American Society of Gene & Cell Therapy
references
1. Aiuti, A, Cattaneo, F, Galimberti, S, Benninghoff, U, Cassani, B, Callegaro, L et al. (2009). Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N Engl J Med 360: 447–458. 2. Biffi, A, Montini, E, Lorioli, L, Cesani, M, Fumagalli, F, Plati, T et al. (2013). Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science 341: 1233158. 3. Naldini, L (2011). Ex vivo gene transfer and correction for cell-based therapies. Nat Rev Genet 12: 301–315. 4. Chuah, MK, Evens, H and VandenDriessche, T (2013). Gene therapy for hemophilia. J Thromb Haemost 11 (suppl. 1): 99–110. 5. Moayeri, M, Ramezani, A, Morgan, RA, Hawley, TS and Hawley, RG (2004). Sustained phenotypic correc-
commentary
tion of hemophilia A mice following oncoretroviralmediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells. Mol Ther 10: 892–902. 6. Follenzi, A, Raut, S, Merlin, S, Sarkar, R and Gupta, S (2012). Role of bone marrow transplantation for correcting hemophilia A in mice. Blood 119: 5532–5542. 7. Shi, Q, Wilcox, DA, Fahs, SA, Fang, J, Johnson, BD, Du, LM et al. (2007). Lentivirus-mediated platelet-derived factor VIII gene therapy in murine haemophilia A. J Thromb Haemost 5: 352–361. 8. Kuether, EL, Schroeder, JA, Fahs, SA, Cooley, BC, Chen, Y, Montgomery, RR et al. (2012). Lentivirusmediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity. J Thromb Haemost 10: 1570–1580.
9. Wang, X, Shin, SC, Chiang, AFJ, Khan, I, Pan, D, Rawlings, DJ et al. (2015). Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A. Mol Ther 23: 617–626 10. Agudo, J, Ruzo, A, Kitur, K, Sachidanandam, R, Blander, JM and Brown, BD (2012). A TLR and non-TLR mediated innate response to lentiviruses restricts hepatocyte entry and can be ameliorated by pharmacological blockade. Mol Ther 20: 2257–2267. 11. Wang, CX, Sather, BD, Wang, X, Adair, J, Khan, I, Singh, S et al. (2013). Rapamycin relieves lentiviral vector transduction resistance in human and mouse hematopoietic stem cells. Blood 124: 913–923.
See page 707
Antiobesity Strategy Targets Energy Economy Safeguards Michel Vivaudou1–3 and André Terzic4 doi:10.1038/mt.2015.39
A
fundamental barrier in obesity management is that caloric restriction triggers energy-conserving responses that evolved to prevent body weight loss. ATPsensitive potassium (KATP) channels have been identified as safeguards controlling energy expenditure in skeletal muscles and thereby key factors determining body weight.1 In this issue of Molecular Therapy, Koganti and colleagues report the successful reduction of muscle energy efficiency through targeted intramuscular injections of cell-penetrating vivo-morpholinos to prevent translation of the channel poreforming Kir6.2 subunit.2 In this elegant proof-of-concept study, the authors demonstrate localized reduction of KATP channel expression and function, leading in turn to an increase in activity-related energy consumption, without compromising exercise tolerance. This report opens a new avenue of investigation in targeted 1 Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France; 2Centre National de la Recherche Scientifique, Institut de Biologie Structurale, Grenoble, France; 3CEA, Institut de Biologie Structurale, Grenoble, France; 4Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, USA Correspondence: Michel Vivaudou, Institut de Biologie Structurale, 71 Avenue des Martyrs, 38044 Grenoble, France. E-mail: vivaudou@ ibs.fr or André Terzic, Center for Regenerative Medicine, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA. E-mail: [email protected]
Molecular Therapy vol. 23 no. 4 april 2015
therapies aiming to control weight management. Obesity reflects an imbalance between calorie intake and expenditure. At present, more than 1 billion adults worldwide are considered overweight, underscoring a rampant epidemic.3 The global prevalence of obesity has precipitated a major escalation in comorbidities associated with an increase in overall mortality.4 Beyond intensive counseling and change in lifestyle, diverse strategies targeting weight loss are being pursued.5 Progress is reflected by US Food and Drug Administration approval of new medications for chronic weight management in obese patients, such as lorcaserin and phentermine/topiramate. Yet antiobesity drug therapy has been largely unsuccessful because of lack of efficacy, poor adherence, and adverse effects.6 Elucidating the molecular pathways that underlie the caloric intake–expenditure equilibrium is necessary to inform the selection of promising therapeutic targets. Indeed, a deeper insight into the innate mechanisms regulating appetite, nutrient exposure, and energy balance is warranted to aid in the discovery and future development of next-generation therapies. Stimulation of energy expenditure may be a potent strategy for obesity treatment. KATP channels, expressed at high density in striated muscles and other excitable tissues, are established membrane sensors of ATP/ADP.7–9 It has been postulated that
under conditions of energy deficit, activation of channel complexes would result in protective energy economy, whereas under energy surplus, downregulation of KATP channels would increase thermogenesis.10 As such, KATP channels provide a low-fuel warning that signals muscle fibers to slow down and avoid irreversible energy depletion. Through tight regulation by adenine nucleotides and integration with metabolic pathways, muscle KATP channels seem to sense both static metabolic levels and the dynamics of energy consumption, thus maintaining an optimal balance between lost heat production and useful mechanical work.11 Without functional KATP channels, energy efficiency decreases and muscles burn more calories than normal.1 Thus, weight loss could be achieved without additional exercise, by “simply” reducing the activity of skeletal muscle KATP channels. A pharmacological approach is conceivable, as numerous molecules are known to modulate KATP channels by binding to their regulatory SUR subunit.12 Unfortunately, blockers of KATP channels specific for skeletal muscle do not yet exist and are unlikely to be discovered soon, given that comparable channels (incorporating isoform SUR2) are also found in cardiac and smooth muscle.13 In the new work, Koganti and colleagues2 used an alternative approach of reducing protein levels by leveraging antisense oligonucleotides packaged as a 615
