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Delivering Hematopoietic Stem Cell Gene Therapy Treatments for Neurological Lysosomal Diseases Rebecca J. Holley, Shaun R. Wood, and Brian W. Bigger*
ACS Chem. Neurosci. Downloaded from pubs.acs.org by 5.62.159.224 on 08/24/18. For personal use only.
Stem Cell and Neurotherapies, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, U.K. ABSTRACT: Neurological lysosomal storage diseases are rare, inherited conditions resulting mainly from lysosomal enzyme deficiencies. Current treatments, such as enzyme replacement therapy and hematopoietic stem cell transplantation, fail to effectively treat neurological disease due to insufficient brain delivery of the missing enzyme. Ex vivo gene therapy approaches to overexpress the missing enzyme in hematopoietic stem cells prior to transplant are an emerging technology that has the potential to offer a viable therapy for patients with these debilitating diseases. KEYWORDS: lysosomal storage diseases, enzyme replacement therapy, hematopoietic stem cells, mucopolysaccharidoses, gene therapy
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insufficient enzyme levels achieved in the brain (Figure 1). This could be due to either poor trafficking of donor cells to the sites of interest or insufficient endogenous enzyme secretion from transplanted cells. The former seems less likely given that replacement of resident microglial cells with 20− 50% bone marrow derived macrophages in the brain can be easily tracked in mice within a month following busulfan conditioning. Gene therapy modification of donor hematopoietic stem cells (HSCGT) to overexpress the missing enzyme prior to cell delivery is therefore an exciting treatment avenue for MPS patients where no effective therapy exists. Third generation lentiviral (LV) vectors are the current choice, delivering safe cellular transduction and high-level enzyme expression in a variety of animal models. Clinical testing of HSCGT in the LSD metachromatic leukodystrophy (MLD) provided first evidence of disease stabilization and measurable CNS effect.1 A phase I/II trial in pre/early symptomatic patients using autologous HSCs transduced ex vivo with a LV vector encoding human arylsulfatase A (ARSA) cDNA, led to stable cell engraftment, increased ARSA activity in peripheral blood and CSF, a reduction in CSF sphingolipids, and preservation of motor and cognitive abilities. This sets a precedent for the use of HSCGT in other neurological LSDs. Building on the success of HSCGT in MLD, several genetic engineering approaches have been employed to maximize brain targeting for the treatment of MPS patients (Figure 1). First, the LV vector used in the MLD trial contained a ubiquitous human phosphoglycerate kinase (hPGK) promoter, designed to drive ARSA expression in all cells. The drawback of this promoter is the high ratio of peripheral enzyme expression relative to the brain. Therefore, an improved lentiviral vector was constructed containing the myeloid-specific CD11b promotor to drive high-level expression in myeloid lineages, the main population engrafting in the brain. Using this strategy
ysosomal storage diseases (LSDs) are mainly caused by defective lysosomal hydrolases, resulting in around 70 different inherited diseases with overall incidence of 1:7000 newborns. Although they exhibit diverse clinical manifestations, all result in the abnormal storage of undegraded substrates, compromising cellular function and leading to severe pathology. When the brain is involved, this leads to devastating neurological symptoms. Delivery of functional enzyme to all cells of the body is critical for successful treatment of LSDs. One class of LSD, the mucopolysaccharidoses (MPS), results in the buildup of glycosaminoglycans in the lysosome, with the subtype of MPS disease dictated by which enzyme is missing. Enzyme replacement therapy (ERT), based on intravenous administration of exogenous enzyme, is able to alleviate disease burden in patients, where symptoms are largely restricted to somatic organs. Treatment efficacy relies on a cross-correction mechanism; enzyme is taken up by surrounding defective cells via the mannose-6-phosphate receptor pathway and sorted into the lysosomal pathway, restoring lysosomal function. However, exogenous enzyme cannot cross into the brain, providing no relief from the severe neurodegeneration experienced by the majority of patients with MPSII or MPSIII subtypes. Thus, the development of a single life-long therapy that can treat both neuropathology and somatic disease burden together is ultimately the primary goal for clinicians treating LSD patients. Haematopoietic stem cell transplantation (HSCT) is approved for the treatment of severe MPSI Hurler patients. In these patients, HSCT provides somatic correction as well as delivering sufficient enzyme to the brain to reduce neurological impairment and improve various clinical outcomes. This bodywide correction is achieved first via the scale-up expansion of hematopoietic populations to recapitulate the entire blood system and therefore providing peripheral enzyme and second via the trafficking of myeloid/macrophage populations across the blood−brain barrier, where they efficiently engraft within the brain, replacing resident microglia and secreting functional enzyme. Despite similarities between MPS subtypes, HSCT has proved ineffective in other MPS subtypes, presumably due to © XXXX American Chemical Society
Received: August 8, 2018 Accepted: August 9, 2018
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DOI: 10.1021/acschemneuro.8b00408 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience
Figure 1. Improving brain-targeting of lysosomal enzymes following hematopoietic stem cell gene therapy. A: HSCT results in complete recapitulation of the blood-system by donor-derived cells resulting in enzyme release (red arrows) into the blood and enzyme uptake (blue arrows) and correction of somatic tissues. Donor-derived macrophages can also cross the blood-brain barrier and engraft within the brain tissue enabling release of enzyme within the brain. However, poor enzyme expression from donor cells limits correction of the brain. B: HSCGT strategies to improve delivery of the missing enzyme to the brain. Enzyme levels can be increased by overexpressing the missing enzyme using modified lentiviral gene therapy vectors. Addition of additional sequences, e.g. ApoEII peptide, can increase enzyme circulation and/or stability, increasing the enzyme in circulation. Altering the enzyme can also increase cell uptake, cell associate and receptor binding aiding superior cross-correction. Increasing the passage of enzyme or passage of cells across the blood−brain barrier, e.g. by direct delivery of stem cells into the brain, will increase the amount of enzyme in the brain.
the target gene was modified by fusing IDS with the lowdensity lipoprotein receptor/heparan-binding domain of human apolipoprotein E as a tandem repeat (LV.CD11b. IDS.ApoEII).4 Direct comparison of LV.CD11b.IDS and LV.CD11b.IDS.ApoEII transplanted mice identified significantly enhanced neurological correction with addition of the ApoEII peptide, with similar vector copies per cell. Although only marginal differences in brain enzyme could be detected (3.4 versus 3.7% of WT), superior correction of the brain with LV.CD11b.IDS.ApoEII was attributed to multiple factors; ApoEII-addition led to 3-fold increases in IDS activity in the plasma, increased enzyme activity per unit protein in the plasma, and increased uptake and transcytosis of the modified enzyme by brain endothelial cells. Uptake was dependent on mannose-6-phosphate, ApoEI and heparan sulfate dependent pathways, with the added advantage that the ApoEII and heparan sulfate binding sites overlap, thus aiding IDS-ApoEII binding and enzyme uptake. Vector design incorporating these adaptations will likely improve HSCGT efficacy in the clinic to provide an effective way of ensuring sufficient provision of enzyme to the brain. Another approach is to bypass hematopoietic repopulation of the bone marrow and trafficking of cells to the brain by instead delivering cells directly into the brain.5 Shown in an
in MPSIIIA animals, HSCGT following transduction with LV.CD11b.SGSH resulted in significantly improved enzyme levels in the brain over use of the PGK promoter; levels were 11% of wild-type following LV.CD11b.SGSH HSCGT versus 7% with LV.PGK.SGSH HSCGT, without significant loss of expression in the periphery (473 vs 576%, respectively).2 Enhanced brain correction was subsequently seen with LV.CD11b.SGSH HSCGT with normalization of MPSIIIA behavior, brain heparan sulfate levels, and neuropathology markers. Conversely, LV.PGK.SGSH only partly corrected neuropathology but not behavior, thus demonstrating improvements in brain specificity with the CD11b promotor. Further efficacy of this vector backbone was shown in a murine model of MPSIIIB with LV.CD11b.NAGLU HSCGT treatment achieving brain NAGLU enzyme levels of 13% of wild-type, with full correction of behavior abnormalities, brain heparan sulfate storage, and neuroinflammatory and neuropathology markers.3 Having improved the expression ratio of brain to peripheral enzyme in engrafting cells, a second approach was sought to determine whether the enzyme itself could be modified to cross the blood−brain barrier, increasing the donor-produced enzyme trafficking to the brain from the periphery. Using a similar myeloid-specific HSCGT strategy in MPSII animals, B
DOI: 10.1021/acschemneuro.8b00408 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience
(3) Holley, R. J., Ellison, S. M., Fil, D., O’Leary, C., McDermott, J., Senthivel, N., Langford-Smith, A. W. W., Wilkinson, F. L., D’Souza, Z., Parker, H., Liao, A., Rowlston, S., Gleitz, H. F. E., Kan, S.-H., Dickson, P. I., and Bigger, B. W. (2018) Brain 141, 99. (4) Gleitz, H. F., Liao, A. Y., Cook, J. R., Rowlston, S. F., Forte, G. M., D’Souza, Z., O’Leary, C., Holley, R. J., and Bigger, B. W. (2018) Brain-targeted stem cell gene therapy corrects mucopolysaccharidosis type II via multiple mechanism. EMBO Mol. Med. 10, e8730. (5) Capotondo, A., Milazzo, R., Garcia-Manteiga, J. M., Cavalca, E., Montepeloso, A., Garrison, B. S., Peviani, M., Rossi, D. J., and Biffi, A. (2017) Sci. Adv. 3 (12), e1701211.
MLD model, intracerebroventricular delivery of LV.ARSA HSCs enabled rapid and robust engraftment of microglial-like cells in the brain enabling efficient brain delivery of ARSA. A drawback of this approach is the lack of somatic correction, however the authors also noted that additional benefit could be achieved if cells were delivered via both intracerebroventrical and intravenous routes, although this will require clinical validation. Reducing the toxicity of conditioning regimens will certainly broaden the scope of HSCGT, but we should be mindful that brain trafficking relies on replacement of microglia, currently mediated by busulfan. Although antibody conditioning protocols are in trial, these may not allow for effective repopulation of the brain. Choosing the right outcome measures ahead of clinical trial is challenging. Several recent gene therapy trials have demonstrated reductions in biochemical CSF based markers without significant improvement in behavioral outcomes, calling into question the validity of these measures. In MPS patients with significant neurological disease, the prioritizing of neurological outcomes over somatic outcomes as clinical end points is preferable. This would need to involve the use of both behavioral (hyperactivity, social) and cognitive measures (memory, language skills). Natural history studies are crucial as, given the severity of the condition and the low number of patients, using placebo treatments is not appropriate. Additionally, it may be important to run clinical trials in these conditions for longer periods due to the chronic and varied progression of disease that occurs in several LSDs. The most important consideration of any gene therapy treatment is how to define a successful treatment. Treating patients early is likely to be essential, necessitating animal studies to determine at what stage the risks of transplant outweigh the usefulness of HSCGT. Ultimately, an affordable, transformative treatment should be the goal, but we must manage expectations for what gene therapy can deliver, which is likely to be a change in phenotype rather than complete cure of disease.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Brian W. Bigger: 0000-0002-9708-1112 Notes
The authors declare the following competing financial interest(s): B.W.B. is shareholder in Orchard Therapeutics and has licensed stem cell gene therapy programs for MPSIIIA and MPSIIIB to Orchard Therapeutics. B.W.B. is named as a coinventor on a patent application (GB1701968.8) for the use of IDS.ApoEII for the treatment of MPS II.
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REFERENCES
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DOI: 10.1021/acschemneuro.8b00408 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
