Gene Therapy for ALD and AMN

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1 Gene Therapy for ALD and AMN May 5 th, 2018 ALD Life Meeting Florian Eichler, MD Massachusetts General Hospital Department of Neurology 12:05 Dr Florian Eichler- Research on gene therapy for ALD and AMN 1

2 Disclosures PI of ex vivo lentiviral gene therapy trial in cerebral adrenoleukodystrophy sponsored by bluebird bio PI of L-serine trial in HSAN1 sponsored by FDA Orphan Disease Group, R01 FD NINDS: R01 NS072446, R01 NS Consultant to Covance, Minoryx Therapeutics and Shire Consultant to Third Rock Ventures on rare neurological disorders Founder of SwanBio Therapeutics 2

3 Overview Delivery methods for CNS gene therapy X-linked adrenoleukodystrophy: phenotypes and pathogenesis Ex vivo lentiviral gene therapy for childhood cerebral ALD In vivo AAV-mediated gene therapy for adrenomyeloneuropathy Gene therapy can take place in two forms: in vivo and ex vivo. In vivo gene therapy involves direct delivery into the brain, spinal fluid or blood. Ex vivo therapy involves extraction of genes which are then treated outside of the body before being reinserted. 3

4 Viral Vectors for CNS Gene Therapy Adeno-associated virus (AAV) 4.7 kb non-enveloped DNA viruses specificity of AAV depends on its classification effectiveness depends on: brain region target cell type species Lentiviral (LVs) 8 kb enveloped RNA viruses ability to integrate into host s DNA stably transfers genetic material to immature cells (particularly those that can develop into all types of blood cells) There are two types of viral vectors to consider Adeno-Associated Virus (AAV)- smaller viral vectors Lentiviral (LV)- larger viral vectors 4

5 Gene Therapy Strategies in vivo gene therapy AAV-mediated ex vivo gene therapy Lentivirusmediated 5

6 IN VIVO Delivery of Recombinant Vectors Usually delivered locally: into brain, into spinal fluid, into blood In some diseases vulnerable brain regions can be targeted, in others transfer from one cell to another can occur (cross-correction possible) 6

7 EX VIVO Gene Therapy for CNS Disease Usually lentiviral transfection of hematopoetic stem cells purified from the blood or bone marrow Renewal of microglial compartment expressing therapeutic protein after hematopoetic reconstitution 7

8 X-linked Adrenoleukodystrophy (X-ALD) Adrenal dysfunction Acute brain inflammatory demyelination Chronic spinal cord axonal degeneration ABCD1 peroxisomal half-transporter(aldp) 1300 mutations in ABCD1 (nonsense, missense, frame shift, microdeletion) Incidence: hemizygotes plus heterozygotes 1:16,800 8

9 Neurological Phenotypes of X-ALD Cerebral ALD(CALD) most severe phenotype (most happened in childhood) Progressive cerebral demyelination with an inflammatory response in the white matter (WM) of the brain NO available mouse model Adrenomyeloneuropathy (AMN) most common phenotype Progressive axonal degeneration and secondary demyelination in the spinal cord X-ALD mouse model develop AMN phenotype at old age ALD is a single-gene disorder with 2 discrete manifestations: Cerebral ALD, mostly affecting 4-10 year olds, although those with the ALD gene continue to have a 20% risk of cerebral ALD in their 20s AMN, taking place after puberty and affecting the adrenal glands. This is the most common form of ALD Depending on the manifestation of ALD, a different approach to gene therapy should be taken, as these are essentially different diseases, rather than being on a spectrum of symptoms. 9

10 Phenotypic Variability in X-ALD 10

11 Neuropathology in X-ALD inflammatory demyelination of brain (CALD) symmetric confluent macrophage and lymphocytic infiltration predominantly parieto-occipital chronic axonal degeneration of spinal cord (AMN) symmetric dorsal columns and corticospinal tract Cerebral ALD can be seen on an MRI scan as an inflammation in the brain, while AMN is difficult to see on an MRI scan and does not lead to any disruption of the bloodbrain barrier. 11

12 Measuring neurologic progression in CALD Functional measures Neurological Function Score 1 Neurological Function Score (NFS) 15 domains Total possible score of 25 Component score Component score Hearing/auditory processing problems Aphasia/apraxia 1 1 Walking difficulties/ spasticity Spastic gait (need assistance) Loss of communication 3 Wheelchair dependence Vision impairment 1 No voluntary movement 3 Major Functional Disabilities (MFDs) 6 most serious symptoms Cortical blindness 2 Episodes of incontinence 1 Swallowing dysfunctions 2 Total incontinence 2 Tube feeding 2 Non-febrile seizures 1 Running difficulties 1 Possible Total Moser et al. Neuropediatrics 2000;31(5):

13 Measuring neurologic progression in CALD Imaging measures Loes MRI severity score 1 : measurement of white matter changes by degree and extent of pathological hyperintense regions (0-34) Gadolinium enhancement: indicator of active inflammation in untreated patients (+/-) Loes score = 1 Loes score = 15 GdE+ 1. Loes et al. AJNR Am J Neuroradiol 1994;15(9):

14 Survival probability Allogeneic bone marrow transplant (BMT) to treat a neurodegenerative disease BMT with donor cells dramatically improves survival Microglial apoptosis in perilesional white matter of CALD HCT treated untreated Eichler et al. Ann Neurol 2008; 63: Bone-marrow derived monocytes enter CNS, differentiate into microglia expressing normal ALDP Years from first abnormal MRI Mahmood et al. Lancet Neurol 2007; 6: marrow HSCs myeloid precursor monocyte microglia Early bone marrow transplants are highly effective, with gene therapy working in a similar way but using the patient s own cells rather than those of a donor. It takes some time after these procedures for patients to stabilise, hence the need for early intervention. 14

15 STARBEAM: Open-label, single-arm phase 2/3 study of Lenti-D in CALD Key enrollment criteria: Age 17 years, evidence of active CALD (GdE+) with early disease (Loes score , NFS 1), no matched sibling donor for BMT Primary outcome: Proportion of patients with no MFDs at 24 months Secondary outcomes: Changes in Loes score and NFS, GdE+ resolution Key safety parameters: Adverse events, detection of replication-competent lentivirus (RCL), insertional oncogenesis Covered by Dr Chiesa s presentation 15

16 STARBEAM treatment protocol overview Covered by Dr Chiesa s presentation 16

17 STARBEAM subject and cellular product characteristics N=18 consented (formally screened) and N=17 infused patients Enrolled n=18 Stem Cell Harvest (apheresis) n=17 Treated n=17 17 subjects treated between October 2013 and July 2015 Data as of August 25, 2017: Total of 21 patients treated Median 30.2 months (min, max [1,46 months]) 4 patients with < 4 months of follow-up Ineligible n=1 (Loes score >9) Parameter Baseline Characteristics Median (range) Age at consent (years) 6.0 (4-13) Time from CALD diagnosis to treatment (months) Time from enrollment to treatment (months) 6.8 ( ) 2.2 ( ) Baseline Loes score 2.0 ( ) Baseline NFS score 0 (0-0) MFDs present at baseline None Covered by Dr Chiesa s presentation 17

18 N F S STARBEAM: 16/17 patients stabilized in their NFS 1 Neurologic Function Scores STARBEAM study NFS Progression in Untreated Patients Historical controls (n=13), Study ALD months since treatment m o n t h s s i n c e f i r s t G d E + s c a n 1. Stable NFS: change of <3 points and an absolute NFS GdE+ untreated subjects regardless of stage of disease with more than 1 NFS score reported (N=13), from time of first GdE+ MRI. Most recent score within 1 year prior to HCT reported as screening score. bluebird bio study ALD-101, manuscript in preparation. Data as of August 25,

19 N F S STARBEAM: 16/17 patients stabilized in their NFS 1 Neurologic Function Scores STARBEAM study NFS Progression in BMT-Treated Patients Historical controls eligible for ALD-102 (n=27), Study ALD months since treatment m o n t h s f r o m t r a n s p l a n t 1. Stable NFS: change of <3 points and an absolute NFS GdE+ HCT-treated (N=27) subjects with Loes score 9 & NFS 1, from time of HCT, all donor sources. Most recent score within 1 year prior to HCT reported as screening score. bluebird bio study ALD-101, manuscript in preparation. 19

20 Neuroimaging outcomes demonstrate halting of disease progression after Lenti-D treatment Subject 2001: first patient treated in STARBEAM pre treatment 1 year after Lenti-D 2 years after Lenti-D Representative untreated patient T1 Post Flair Data as of August 25, 2017 Loes score = 2 Loes score = 3 Loes score = 2 20

21 Subject ID Gadolinium enhancement resolved in 11/15 patients by 24 months GdE- GdE+ Data as of August 25, GdE status (+/-) assigned by central MRI reader blinded to subject and time point 21

22 V C N ( v e c t o r c o p i e s / d i p l o i d g e n o m e ) % C D 1 4 c e l l s p o s i t i v e f o r A L D P e x p r e s s i o n Persistence of vector-marked cells and ALDP expression Vector Copy Number in Peripheral Blood ALD Protein Expression in CD14+ Cells Median, IQR, min, max M 2 M 3 M 6 M 9 M 1 2 M 1 5 M 1 8 M 2 4 M Months since treatment T i m e s i n c e L e n t i - D d r u g p r o d u c t i n f u s i o n ( m o n t h s ) T i m e s i n c e L e n t i - D d r u g p r o d u c t i n f u s i o n ( m o n t h s ) N = VCN in peripheral whole blood ranged from 0.1 to 1.55 at month 24 (N=14) and at month 36 (N=3) All samples have detectable VCN and ALDP expression at last follow-up Data as of August 25,

23 Ex Vivo Gene Therapy for X-ALD Milestones: First Trial of Single Gene Addition in X-ALD Encouraging Efficacy Data (no Major Functional Disabilities to date) Reassuring Safety Profile (no deaths, no clonal dominance) Limitations: Delays in engraftment time = brain Adverse events consistent with a suppressed bone marrow HSCT X-ALD patients also develop AMN (Van Geel et al, 2015) AMN requires broad delivery to the entire spinal cord/peripheral nerve Are there other approaches for AMN? 23

24 Select axons need glial peroxisomes Corticospinal tract Dorsal columns adrenomyeloneuropathy is a non-cell-autonomous process of selective vulnerability AMN requires a different approach, such as targeting the long tracts in the spinal cord, and the cells protecting these tracts. The approach needed for AMN should be similar to that taken for other spinal cord issues, such as Spinal Muscular Atrophy (SMA), in which there have been major advances in the past year. 24

25 Adeno-associated virus (AAV): a direct transduction approach AAV: A small DNA virus which infects humans and some other primate species Safety: does not cause disease and is relatively non-inflammatory Serotypes:AAV1,2,3,4,5,6,8,9, 10(cross BBB) Fast, robust transgene expression in the CNS AAV mediated gene therapy: > 90 clinical trials using AAV vectors (inherited diseases, CNS disorders, heart failure), indicating safety April 2016: 15 infants with spinal muscular atrophy treated successfully with scaav9.cb.smn 25

26 AAV#to#target#neurodegeneration#in#the#spinal#cord target in the Adrenomyeloneuropathy Dorsal'root'ganglionroot Astrocytes Microglia Endothelial'cells Corticospinal,tracttract Dorsal,columns Spinal'Muscular'Atrophy Lower'motor'neuron 26

27 Vector construct of recombinant AAV9 carrying human ABCD1 Chimeric intron b-actin exon Chicken beta-actin promoter CMV IE enhancer ITR HindIII (1) ABCD1 Yi Gong ColE1 ori paav-cba-habcd1-w 7467 bp Xho I (2245) AmpR WPRE SV40 polya Casey Maguire F1 ORI ITR BGH polya 27

28 AAV9-mediated gene delivery of human ABCD1 is possible Peroxisome Localization Brain Spinal cord intracerebroventricular intravenous Gong, et.al., 2015, Molecular Therapy ABCD1 delivered to CNS either directly into the blood or into the spinal fluid via brain catheter In the dish it delivers into the correct cell compartment (peroxisome) Lead to a reduction in very long chain fatty acids 28

29 Hind limb reflex extension score Abcd1-/- mouse characteristic: excessive hind limb clasping Hind Limb Reflex Extension Score score activity 0 paralysis of both hind limbs 0.5 paralysis of one hind limb 1 clasping of hind limbs Extension Flexion Clasp * * * * * WT Abcd1-/- 1.5 alternating clasping and flexion of hind limbs 2 flexion of hind limbs 2.5 alternating flexion and extension of hind limbs in an angle < 90 3 extension of hind limbs in an angle < 90 Starts around 15months of age months old High score indicates less clasping behavior 3.5 alternating extension of hind limbs in an angle < 90 and less then 90 4 extension of hind limbs in an angle of more than 90 degrees. Tests have been done on mice experiencing changes in motor performance similar to AMN, shown in clasping motions. 29

30 Hind limb reflex extension score Hind limb reflex extension score Improved motor performance after AAV9-ABCD1 injection via ICV months age ICV: 10E11gc/mouse treated at 5 months * # * # * * * WT Abcd1-/- Abcd1-/-ICV 4 WT Abcd1-/- Abcd1-/- ICV treated at 13 months 2 2 ** months age months age Extension Flexion Clasp These symptoms can be relieved when treated both early and late, using a pump to directly insert treatment into the spine. 30

31 Aims of pursuing intrathecal osmotic pump 1. Optimize AAV9-ABCD1 delivery for AMN treatment purpose 2. Increase spinal cord targeting 3. Reduce peripheral leakage L4-L5 Entry site 31

32 GFP pixel quantification(% area) Is pump delivery of AAV9 more efficient in reaching the spinal cord than bone marrow transplantation? 15 Cervical Thoracic Lumbar AAV9-IT pump BMT Difficult comparison: Ex vivo versus in vivo After 2 weeks (AAV9) versus 3 months (BMT) Dose comparison (3x10 11 gc versus 20 mill donor BM cells) 32

33 Less leakage of protein into the circulation after delivery to spinal fluid via pump Comparison with bolus injection Comparison with intravenous delivery 33

34 C26:0 level(nmol/mg protein) habcd1 protein expression in spinal cord, brain and DRG after intrathecal pump delivery versus bolus injection TOPRO3 (Nucleus) ABCD1 CD31 ABCD1 GFAP 12 *** spinal cord ### SC SC 8 ### TOPRO3 (Nucleus) ABCD1 IBA1 TOPRO3 (Nucleus) ABCD1 4 0 WT PBS AAV9-ABCD1 AAV9-ABCD1 pump bolus Abcd1-/- SC DRG A multimodal approach may be possible in the future, utilising different kinds of gene therapy. 34

35 Conclusion 1. Reassuring safety data and encouraging efficacy data of first GT trial in ALD (and overall in the leukodystrophies) Lenti-D gene therapy may offer an alternative to allo-hsct in patients with early cerebral disease, particularly without matched sibling donor 2. Specific phenotypes within an individual leukodystrophy require different approaches (ex vivo versus in vivo GT) 3. The timing of treatment is critical for Cerebral ALD : When early inflammation visible on brain MRI Before lesion too extensive 35

36 Conclusion 1. Within each phenotype the target structures and cells are critical: In cerebral ALD, microglial pathology is prominent and correction of bone marrow cells contributes to brain health. In AMN, no disruption of the blood brain barrier is present and AAVmediated gene transfer into spinal fluid leads to widespread expression. 2. In the future, a multimodal approach will likely be required over time 3. Route and overall approach to gene delivery makes a difference 36

37 Massachusetts General Hospital (Eichler lab) Yi Gong Casey Maguire Patricia Musolino Jun Byungkyu Dakai Mu JiaQian Ren Ann Moser Rene Kok Leblang Charitable Foundation NIH (R21 NINDS, R01 NINDS) Pennsylvania Orphan Disease Center Acknowledgements Boston Children s Hospital David A. Williams Christine Duncan University of Minnesota Gerald V. Raymond Paul J. Orchard Weston P. Miller Troy C. Lund Fundagen SA, Buenos Aires Hernan Amartino Hôpital Universitaire Robert Debré and Hôpital Saint-Louis, Paris André Baruchel Jean-Hugues Dalle Jerome Larghero UCLA Raman Sankar Donald B. Kohn Satiro De Oliveira Ami J. Shah Women s and Children s Hospital, South Adelaide Nicholas J.C. Smith Drago Bratkovic Great Ormond Street Hospital, University College London Adrian Thrasher H. Bobby Gaspar Paul Gissen bluebird bio, Cambridge Asif M. Paker Tara O Meara Esther Shamir INSERM & Hôpital Bicêtre, Paris Patrick Aubourg 37