In The Nameof GOD

New era in treatment of IMD

Peymaneh Sarkhail M.D.

Pediatric Endocrinologist & Metabolic Consultant

Introduction

• Inborn errors of metabolism are collectively common, affectingapproximately 1 in every 500-1500 newborns.

• Close to 10% of currently annotated human genes encode enzymes orenzyme subunits, explaining the prevalence and clinical importance ofmetabolic disease.

• Disease phenotypes are frequently severe, and in many instances,inadequately managed by current therapies.

• These include Metabolic manipulation (like diet or drugs), enzymereplacement (ERT), Substrate reduction therapy (SRT) and chaperon therapyin some disorders.

• The new modalities in treatment of IMD include organ transplantation, celltherapy , and Gene therapy

Gene Therapy (2008) 15, 831–839

Therapy of IMD

Organs Liver Tyrosinemia

UCD

MMA

Wilson

α1-Anti trypsin

Kidney Fabry Disease

Cystinosis

MMA

Cells HSCT SCID

ALD

MPSs

Gaucher-II,III

NCL

MSC MLD

MPSs

Some LSDs

• Since its inception in 1963, Orthotopic liver transplantation has been theultimate solution for most patients refractory to palliative or solution basedmanagement .

• Among them, urea cycle disorders (UCDs) are the most common indication fortransplantation, accounting for 25.6% of the cases

• This is followed by alpha 1 antitrypsin deficiency (19.7%)

• Cystic fibrosis (10%)

• Wilson disease (7.6%)

• Maple syrup urine disease (MSUD) (6.5%)

• Tyrosinemia (7.4%)

• Glycogen storage disease (GSD) (5.2%)

• Other disorders (17.2%)

Liver Transplantation in IMD

Pediatr Transplant. 2016 September ; 20(6): 756–769.

Exp Hematol. 2008 June ; 36(6): 716–725.

Liver Transplantation in IMD

Pediatr Transplant. 2016 September ; 20(6): 756–769.

• Importantly, even in cases where LT is non-curative, it can provide patients andtheir families with substantial benefit by reducing the risk of acute metabolicdecompensation or stringent dietary restriction, thereby significantly improvingtheir quality of life

• It is important to note that some genes that have significant extrahepaticexpression will still be effectively cured by LT as the residual abnormalmetabolites generated by the rest of the body can be efficiently cleared by thedonor liver.

• An example of such a disorder is MSUD, which is caused by mutations in any ofseveral genes encoding a multi-subunit enzyme complex responsible forbranched chain amino acid disposal.

• These genes are highly expressed in liver but are also expressed in muscle

Hepatocyte transplantation in IMD

• The easiest disorders to correct are those which respond well to replacementof a relatively small portion of liver by cells bearing functional gene.

• In such cases, terminally differentiated hepatocytes may be useful for cell-based enzyme replacement

• Crigler-Najjar Syndrome, as one of the earlier records of such an experimentaltreatment.

• By infused normal hepatocytes via the portal vein, plasma bilirubinconcentrations stably decreased, ameliorating clinical symptoms of the disease

• The mouse model for hereditary tyrosinemia type 1 represents one disorder inwhich the transplanted hepatocytes possess a substantial proliferativedisadvantage.

• Unable to produce fumaryl acetoacetate hydrolase (FAH), resident hepatocytessuccumb to the toxicity of tyrosine catabolites

Exp Hematol. 2008 June ; 36(6): 716–725.

Hematopoietic Cell Therapy for IMD

• The delivery of enzyme may be achieved by exogenous enzyme administration,via allogeneic hematopoietic stem cell transplantation (HSCT).

• As microglial cells, which are hematopoietically derived, engraft within the brain,HSCT could prove efficacious in continuous delivery of enzyme within the CNS

• It is an effective alternative therapy for ERT in some MPS like MPS type I, II, IV,and VI.

• Little published experience exists on the efficacy of HSCT in MPS VII, butresponses have been described

• Similarly, early results with HSCT using allogeneic bone marrow were notencouraging for Sanfilippo syndrome (MPS III).

J Pediatr 2007;151:340-6

Hematopoietic Cell Therapy for IMD

• Transplantation early in the course of cerebral ALD has been shown tostabilize the disease, but in more advanced patients the outcome is poor.

• In globoid cell leukodystrophy (GLD), allogeneic HSCT has proven capable ofarresting disease progression in late onset form but in symptomatic infantsthe results of HSCT have been poor.

• HSCT could rapidly normalize arylsulfatase A level, and disease stabilizationmay be achieved in many patients with the adult and juvenile forms of MLD.

• However, in younger patients with late infantile MLD, progressivedeterioration with spasticity and ataxia is often observed in spite of HSCT

J Pediatr 2007;151:340-6

Hematopoietic Cell Therapy for IMD

• Outcome reports evaluating allogeneic HSCT as treatment of otherinherited metabolic disorders are few.

• In Wolman disease, early HSCT restores normal plasma levels of acidlipase, with resolution of organomegaly and correction of malabsorption

• In I-cell disease (mucolipidosis-II) HSCT has been reported to improve thiscondition

• In alpha-mannosidosis, stabilization of neurocognitive variables andpossibly musculoskeletal complications has been observed.

• To date there is no convincing evidence HSCT provides benefit in thegroup of disorders termed gangliosidoses (Tay-Sachs, Sandhoff, GM1gangliosidosis), and Niemann-Pick .

J Pediatr 2007;151:340-6

• Ratko TA, Belinson SE, Brown HM, et al. AHRQ Comparative Effectiveness Review No. 48. Available at www.effectivehealthcare.ahrq.gov/stem-cell-children.cfm.

Alternative cell sources

• The ability of HSCT to stabilize CNS disease in inborn errors of metabolism isassumed to result from the migration of donor derived monocytes and theirprecursors into the brain to become microglia, leading to enzymatic cross-correction of the host neural cells.

• However, administration of these hematopoietic cell populations has failed toachieve metabolic correction in many tissues, such as cardiac valves, peripheralnerve and cartilage.

• In addition, it appears there is limited delivery across the blood-brain barrier, basedon the observation that patients may continue to deteriorate despite fullengraftment.

• Other allogeneic cell– based approaches targeting these tissues have the potentialto greatly improve therapy.

• Ideally, a multilineage stem cell would home to the site of injury after infusion anddifferentiate into the appropriate cell type.

J Pediatr 2007;151:340-6

Alternative cell sources

• Peripheral blood stem cells, and unrelated cord blood grafts contain suchmultipotential cells.

• Mesenchymal stem cells (MSC) and related multipotent adult progenitorcells (MAPC) can be isolated from bone marrow.

• Because of their multilineage potential, including the capacity todifferentiate into non-hematopoietic mesenchymal cells (hepatocytes,adipocytes, myocytes).

• Both MSC and MAPC may prove important candidates for non-hematopoietic, allogeneic cell therapy.

• In patients with MLD, infused allogeneic MSC was associated withimprovement in nerve conduction velocities.

J Pediatr 2007;151:340-6

Alternative cell sources

• The neurologic aspects of LSD could be treated by infusion of neural or glialprogenitor cells, derived either from neuronal stem cells or from MAPC.

• The blood-brain barrier limits penetration of intravenously administeredenzyme, and likely alters cell localization as well

• Alternative strategies for delivery of MSC, MAPC, or other cell types into thebrain include intrathecal or intraventricular injection systemic intravenousadministration simultaneously with HSCT

• In the context of HSCT, non-hematopoietic progenitor cells may enhanceengraftment, alter immunologic responses and prevent or control GVHD.

• In addition, MSC or MAPC offer the potential for correction of othercomplications poorly treated by HSCT, such as orthopedic and valvular diseasein MPS disorders.

J Pediatr 2007;151:340-6

Gene therapy in IMD

• In the early 1970s, the concept of gene therapy became a reality when anexogenous gene was delivered to a mammalian cell resulting in proteinexpression

• The first clinical gene therapy trial was performed at the National Institutesof Health (NIH) in 1990 to treat severe combined immunodeficiency causedby adenosine deaminase deficiency (ADA-SCID).

• In 1999, a gene therapy clinical trial for ornithine transcarbamylasedeficiency (OTC) resulted in the death of patient, which was caused by asevere immune response to the therapeutic adenoviral vector (AAV).

• Then in 2003, it was reported that patients in a gene therapy trial for X-linked severe combined immunodeficiency (SCID-X1) developed a T-cellleukemia caused by insertional mutagenesis of the vector

• These severe adverse events retarded the progress of the enthusiasm forgene therapy as treatment for genetic disease

Translational Science of Rare Diseases 1 (2016) 73–89

Gene therapy in IMD

• To date over 40 metabolic disease phenotypes have been investigated in smallanimal studies with complete phenotype correction being achieved in asubstantial proportion

• Four metabolic disease phenotypes have reached phase I or I/II trials withone, targeting lipoprotein lipase deficiency, showing exciting early evidenceof efficacy

• In 2012 Glybera®, an adeno-associated viral gene therapy vector, wasapproved to treat lipoprotein lipase deficiency by the European Commission .

• To date in the United States, there have been no approved gene therapytreatments.

Translational Science of Rare Diseases 1 (2016) 73–89

Gene Therapy (2008) 15, 831–839

Gene Therapy in IMD

• Two general strategies are used to deliver genes tospecific tissues.

• 1) Ex vivo transfer:

• Involves removing cells from a patient, introducingappropriate genes, and transplanting the geneticallyreconstituted cells into the individual.

• 2) In vivo transfer:

• Involves introducing the gene directly into the affectedtissue,

• Both have been used to transfer genes to hepatocytes,whereas introduction of genes into cells derived fromthe bone marrow has only been achieved using ex vivotransfer

Translational Science of Rare Diseases 1 (2016) 73–89

Gene therapy in IMD

• Ideal disease candidates for in vivo gene therapy are disorders that can beentirely corrected by delivery of therapeutic genes to hepatocytes

• In contrast, metabolic diseases presenting with multisystemic involvementare more challenging to treat because they require transduction of multipletissues for disease correction.

• A thorough understanding of disease pathogenesis permits prediction ofdisorders amenable to correction by liver directed gene therapy, which iscritical for clinical development of gene therapy.

• Clinical experience with liver transplantation is extremely useful to predictthe outcomes of liver-directed gene therapy

• Every disease treated by liver transplantation is amenable to correction bygene therapy

HUMAN GENE THERAPY, 2015 ;26:186–192

Gene therapy in IMD; LSD

• The exciting potential of AAV-mediated gene delivery to meet thischallenge is substantiated by data generated in over 40 studies in animalmodels involving at least 15 different LSD phenotypes.

• For most LSDs, however, the presence of systemic and CNS pathologymakes successful treatment by gene therapy even more challenging.

• This is illustrated by studies in mice with MPS VII (Sly syndrome), recentstudies have focused on CNS and ocular manifestations and systemicdelivery has not yet been revisited with newer capsid serotypes

• Studies in the CNS have included direct stereotactic delivery, andintraventricular and intrathecal injection.

Gene Therapy (2008) 15, 831–839

Gene therapy in IMD; amino acid disorders

• Specific examples that have been investigated as therapeutic targets using rAAVinclude: OTC deficiency, PKU, homocytinuria, MMA, and Canavan disease,

• The earliest of these exploited adenovirus-mediated gene transfer in OTC deficientmice, with phenotype correction first reported in 1996

• this early pre-clinical success soon led to an ill-fated phase 1 clinical trial for partialOTC deficiency in 1999 .

• Whilst the first 17 patients recruited to this vector dose escalation safety studywere treated uneventfully

• Similar to results achieved for OTC deficiency, relatively robust phenotypecorrection has been achieved in mice model of PKU

• Reported reduction in plasma homocysteine levels after systemic AAV-mediatedgene delivery is therefore likely to be the consequence of liver transduction

Gene Therapy (2008) 15, 831–839

J Inherit Metab Dis 2012

DOI 10.1007/s10545-012-9467-0

Gene therapy in IMD; amino acid disorders

• In MMA ,medical management is extremely challenging and the role of liver and liver–kidney transplantation remains controversial because of the systemic nature of theenzyme defect.

• This may be explained by substantial production of methylmalonic acid by other tissues,such as skeletal muscle

• To date AAV2/2 and AAV2/8 vectors encoding cobalamin adenosyltransferase have beenproduced and functionally validated in healthy mice following portal vein delivery.

• There is no effective treatment for Canavan disease.

• Mouse and rat models exist and both have been used to investigate AAV-mediated genedelivery

• Following stereotactic injection regional improvements in characteristic neuropathologyhave been achieved in concert with reduced NAA levels as assessed by MRS.

Gene Therapy (2008) 15, 831–839

Gene therapy in IMD; energy metabolism defects

• Defects in energy metabolism encompass disorders of mitochondrial function, fattyacid oxidation, ketogenesis, ketolysis , and creatine biosynthesis

• Examples of the scenario have been considered as targets for AV mediated genetherapy include:

• Defects in the pyruvate dehydrogenase complex (PDHC), Leber’s hereditary opticneuropathy (LHON) and adenine nucleotide translocator (ATN1) deficiency

• Vectors encoding the E1a subunit for PDHD have been constructed and tested invitro and in vivo, but to date only partial restoration of PDHC activity has beenachieved

• The LOHN disease phenotype has been successfully mimicked in mice byintraocular AAV mediated delivery

• Whether this approach would ameliorate phenotype in vivo remains to be testedand more direct approaches involving delivery of deficient gene products encodedby the mitochondrial genome (mtDNA) have not been reported.

Gene Therapy (2008) 15, 831–839

Gene therapy in IMD; disorders of lipoprotein metabolism

• To date, two conditions in this class have been investigated in animalmodels, familial hypercholesterolemia, due to mutations in the LDLreceptor and lipoprotein lipase deficiency.

• Liver-targeted gene transfer of the human LDL receptor resulted inclose to complete normalization of serum lipid levels in LDL receptordeficient mice .

• Similarly, lipoprotein lipase deficiency has been successfully treated inboth mouse and cat models with clearance of visible hyperlipidemia andplasma triglycerides reduced to near normal levels

Gene Therapy (2008) 15, 831–839

Gene therapy in IMD; carbohydrate disorders

• Disorders of glucose metabolism encompass the GSDs, defects ingluconeogenesis and glucose transport and congenital hyperinsulinism.

• GSD Ia is the only disease in the class to have received attention as a possibletarget for AAV-mediated gene therapy

• Murine and canine models of GSD Ia exist, and both have been used to exploreAAV-mediated phenotype correction.

• In mice, substantial biochemical correction, including abnormal glycogenstorage, has been achieved with vectors bearing serotype 1 or 8 capsids.

• In dogs, consistent reduction in liver glycogen has been achieved withnormalization of fasting glucose, cholesterol, triglycerides and lactic acidreported in one animal

Gene Therapy (2008) 15, 831–839

Gene therapy in IMD; Peroxisomal disorders

• Patients with peroxisomal disorder X-linked adrenoleukodystrophy treatedwith ex vivo lentiviral vector-mediated gene therapy have also exhibitedevidence of clinical benefits

• PH1 is caused by deficiency of the liver peroxisomal enzymealanine:glyoxylate aminotransferase (AGT),

• Combined liver–kidney transplantation is currently the only availabletreatment for most patients with PH1.

• Preclinical proof of concept of efficacy of AAV8 in PH1 mice has beenprovided

HUMAN GENE THERAPY, 2015 ;26:186–192

Gene therapy in other metabolic disorders

• Other metabolic conditions, that have been investigated in animal modelsusing AAV-mediated gene transfer include:

• a-1-antitrypsin deficiency, molybdenum cofactor deficiency, and Crigler–Najjar syndrome,

• In Crigler–Najjar syndrome, intraportal injection vectors showed reducedserum bilirubin levels

• Similarly, liver-targeted delivery of MOCS1 to knockout mice using an AAV-1/2chimeric capsid proved effective in rescuing the neonatal lethal phenotype ofmolybdenum cofactor deficiency and also prevented death

• Finally, therapeutic serum a-1-antitrypsin levels, potentially sufficient toprevent the development of emphysema, have been achieved in micefollowing either intramuscular or intrapleural delivery.

Gene Therapy (2008) 15, 831–839

HUMAN GENE THERAPY, 2015 ;26:186–192

• Genome editing tools are tailorablenucleases with potential to advance thegoals of a gene therapy for IMDs.

• Unlike conventional gene therapy that onlyactivates the expression of a recombinanttransgene, therapeutic genome editingprovides versatile strategies to modify genesto correct an IMD-related deficiency

• The first generation of genome editing toolsare protein-based nucleases, including zincfinger nucleases (ZFNs) and transcriptionactivator-like effector nucleases (TALENs).

YALE JOURNAL OF BIOLOGY AND MEDICINE 90 (2017), pp.553-566.

Genome editing endonucleases in IMD

Genome editing endonucleases in IMD

• Recently, the second generation ofprogrammable genome editing tools knownas RNA-guided nucleases for genome editingin a wide variety of organisms

• This category of tools consists of clusteredregularly interspaced short palindromicrepeats (CRISPR) and CRISPR-associatedprotein 9 (Cas9) system from the bacterialadaptive immune system

• However, these approaches are still far frombeing used in clinical applications becauseof significant issues related to safety anddelivery.

YALE JOURNAL OF BIOLOGY AND MEDICINE 90 (2017), pp.553-566.

Thank you