Cell transplantation to the rescue: Repairing the injured spinal cord. Mary Bartlett Bunge, PhD

Transcription

1 Cell transplantation to the rescue: Repairing the injured spinal cord Mary Bartlett Bunge, PhD Christine E. Lynn Distinguished Professor of Neuroscience Professor of Cell Biology, Neurological Surgery, and Neurology The Miami Project to Cure Paralysis University of Miami Miller School of Medicine

2 Founders (1985) -Barth Green, M.D. -Donald Misner -Beth Roscoe -Nick Buoniconti -Marc Buoniconti Scientific Directors -ÅkeSeiger, M.D., Ph.D. -Richard Bunge, M.D. -W. Dalton Dietrich, Ph.D. Current Staff - 21 basic science faculty - 16 clinical science faculty - 12 management - 18 post-docs - 17 graduate students research staff - 41 support staff - other students (high school, undergrad, medical, residents)

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6 Spinal Cord Injury (SCI) 12,000 new cases / year in the U.S. Approx million Americans with paralysis due to SCI; 5.3 million with paralysis due to some type of CNS injury/disorder Nearly 50%, ages 16-30; 80%,males Higher occurrence in military, Dept of Defense funding MVAs (36.5%), falls (28.5), violence (14)

7 Quality of Life Issues with SCI Muscle paralysis, loss of feeling Reduced pulmonary function Early cardiovascular disease, stroke, DVTs Lack of bowel and bladder control; infection Temperature, blood pressure vary widely Pressure ulcers, osteoporosis Sexual function/fertility impaired Pain long term, 80 % Obesity, diabetes, cognitive decline

8 Cultured cells for CNS repair If we have normal cell types available in culture and if a nervous system disease stems from an absence or deficiency of one of these cell types, then it is an obvious step to transfer cultured cells into the diseased animal to attempt to correct the deficiency. Whereas at present this would not seem practical for many types of neurons, it may be practical for supporting cells (e.g. Schwann or glial). Richard P Bunge, The changing uses of nerve tissue culture in The Nervous System, Donald B Tower, editor in chief, Vol 1 the Basic Neurosciences, 1975.

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10 Nerve cell (neuron)

11 Peripheral nerve

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13 Peripheral nerve Schwann cell myelinated axons

14 Myelin formation by Schwann cells

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16 Albert J. Aguayo, M.D.

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19 Photo of St. Louis Arch

20 Patrick M. Wood, PhD

21 Schwann cell biology in vitro SCs generate a number of ECM components SCs require contact with ECM to differentiate SCs require contact with axons to form basal lamina SCs require basal lamina to form myelin SCs require ascorbic acid to form myelin (Type IV collagen) [Defined medium (N2) supported SC profileration but not differentiation]

22 Schwann cell biology in vitro - 2 Mitogenic signal on axons for SC proliferation (control of SC #) Perineurium formed in cultures of neurons, SCs and fibroblasts Perineurium formed from fibroblasts, not SCs (Lac-Z retrovirus) Circumnavigation by inner lip of SC cytoplasm a part of myelin formation mechanism

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26 Why Schwann cells? Promote axon regeneration/pn Aguayo team; CNS axons regenerated into PN Are readily accessible in PN Produce growth factors, ECM Myelinate axons in CNS, restore activity Can obtain large numbers in culture Can transplant person s own cells Can genetically engineer cells Promising pre-clinical data in multiple species (rats, pigs, primates) ; FDA approval for human SCs

27 Xiao-Ming Xu, M.D., Ph.D. Xu XM, Guénard V, Kleitman N, Bunge MB (1995) Axonal regeneration into Schwann cell-seeded guidance channels grafted into transected adult rat spinal cord. J Comp Neurol 351: Xu XM, Guénard V, Kleitman N, Aebischer P, Bunge MB (1995) A combination of BDNF and NT-3 promotes supraspinal axonal regeneration into Schwann cell grafts in adult rat thoracic spinal cord. Exp Neurol 134: Xu XM, Chen A, Guénard V, Kleitman N, Bunge MB (1997) Bridging Schwann cell transplants promote axonal regeneration from both the rostral and caudal stumps of transected adult rat spinal cord. J Neurocytol 26:1-16.

28 PAN/PVC tube * SC Scanning EM

29 How do we measure outcome? -Behavior: Walking? Pain assessment? -Histology: Lesion volume, overall tissue changes -Transplanted SC survival and number? -Count SC-myelinated axons? Total # axons? -Immunostaining Scar formation? Types of axons in implant, and beyond? Inflammatory response? -Neuroanatomical tracing What spinal and supraspinal neurons responded? What types of axons regenerated into implant? Did these axons leave implant?

30 SC/Matrigel bridge at 30 dpt

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32 Thoracic complete transection model: Schwann cells Axons grow onto SC bridge from both stumps 2000 myelinated axons on bridge 8x more non-myelinated axons/bridge

33 Transplants at 12 wk after contusion injury LV-GFP Dr. Damien Pearse

34 Thoracic contusion model: Schwann cells Reduce cyst formation Protect spinal cord tissue from secondary damage Support axonal growth into SC graft ~5000 SC-myelinated axons in graft Some improvement in walking after paralysis

35 Injured spinal cord tissue environment -1 Excessive EAA (e.g., glutamate) release EAA receptor activation Excessive Ca ++ entry into cells Ca ++ stimulated activation of proteolytic and other degradative enzymes Cytoskeletal protein breakdown Oxygen free radical release leading to membrane damage Cytokine, chemokine release

36 Injured spinal cord tissue environment-2 Many tissue damaging sequelae initiated, leading to secondary damage Substances inhibitory to repair appear Inflammation begins Barrier to axon growth (scar) forms Sustenance for surviving nerve cells cut off Cyst created due to tissue loss, preventing axon regrowth

37 To repair the cord - 1 Halt spread of secondary tissue damage Save as many nerve cells as possible Curb inflammation Reduce scar formation Neutralize inhibitory factors

38 To repair the cord - 2 Awaken nerve cells to regrow axons Provide sustenance to nerve cells Promote axon growth across injury Guide growth to appropriate area Enable formation of connections

39 Combination strategies: SCs + Steroid (used clinically) Variety of growth factors Another cell type (olfactory ensheathing cells) An enzyme (to reduce scar formation) Elevation of a cell signaling molecule, camp Introduction of genes to improve Schwann cell efficacy ALL THESE IMPROVED REPAIR/FUNCTION

40 Combination strategies: SCs + More SC-myelinated axons in graft Increase in regenerated axons from neurons above spinal cord (Distance factor overcome) Exit of regenerated axons from graft into the cord More improvement in walking

41 SCs with added growth factor gene survive better and improve axon growth Control SCs SCs + growth factor

42 Viral Vector/growth factor (GF)- engineered SC study (2007) GF levels signif. increased in cord at 2 dpt, low levels up to 6 wpt GF led to 5X increase in graft vol, SC # GF led to a signif. increase in myel. axons (SC 5,100; LV-GFP SCs 4,600; LV-GFP/GF SCS 26,000) and total axons (~75,000) DβH+ fiber length signif. increased with GF No improvement in walking

43 Combined engineering of Schwann cell implants to secrete neurotrophin and chondroitinase promotes axonal regeneration and locomotion after SCI H Kanno, Y Pressman, A Moody, R Berg, E Muir, J Rogers, H Ozawa, E Itoi, D Pearse, M Bunge

44 Haruo Kanno, M.D., Ph.D.

45 Experiment Adult female Fischer rats ( g) Moderate contusion injury at thoracic 8 level Schwann cell transplantation: 1 week after injury, 1+1 million cells Experimental groups: 1. Vehicle (no cells) (control) 2. green-scs + red-scs (control) 3. green/gf-scs + red-scs 4. green-scs + red/enz-scs 5. green/gf-scs + red/enz-scs

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47 Chondroitinase activity in vivo 2B6 staining GFP mcherry/chabc 2B6 1000μm

48 SC-transplanted spinal cord Control Combination strategy

49 SC myelination in implant # SC myelinated axons / section DMEM/F12 GFP + mcherry GFP/GF+ mcherry GFP + mcherry/chabc GFP/GF + mcherry/chabc * # * 1) DMEM/F12 2) GFP + 3) mcherry GFP/D15A 4) GFP + mcherry + 5) mcherry/ch'ase GFP/D15A + mcherry/ch'ase

50 Combination vs single treatments Further increase in implant compared to GF or ENZ Further increase in SC myel axons compared to ENZ Higher number of responding neurons above cord than with GF or ENZ Higher number of their axons in implant and in cord beyond than with GF or ENZ Improved walking scores Lessened pain in hindpaws

51 Experimental Neurology 1997;148: The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cord Guest JD, Rao A, Olson L, Bunge MB, Bunge RP The Miami Project to Cure Paralysis, The Organ Procurement Team, Department of Neurological Surgery, University of Miami School of Medicine, Miami, Florida Nude athymic rats Human SCs/Matrigel, with or without PAN/PVC channels T8 complete transection, T9-10 cord removed Methylprednisolone

52 Myelinated axons, ~2,000 Propriospinal, sensory, motoneuronal, brainstem (DβH, 5HT) axons in SC bridge Propriospinal and sensory axons re-entered the spinal cord (up to 2.6 mm) Walking scores significantly improved Robust axon growth into SC bridge, some growth beyond and modest improvement in locomotion

53 Human Schwann cells Can be isolated and readily increased in # in culture Up to 10 m SCs, single adult sural nerve biopsy 100 m SCs in 3-4 wk Variation in rate of increase from donor to donor Preparation: Careful dissection of bundles Intro culture medium with forskolin ( camp) and heregulin (mitogenic), 1 wk Overnight incubation in collagenase and dispase; then mechanical dissociation of fascicles by trituration Onto laminin, with forskolin and heregulin (all reduce fibroblasts, leading to 95% SCs or higher)

54 APPROVAL FROM THE FDA FOR OUR FIRST SC TRIAL Phase 1 (for safety) Clinical trial: to inject person s own Schwann cells (from peripheral nerve*) into site of SCI SC number increased in culture for 3-5 wk SCs injected into lesion epicenter *From single adult sural nerve biopsy, 100 m SCs/ 3-4 wk

55 Feasibility for trial? (Many rodent studies over nearly 20 years) Detailed characterization of manufactured hscs Preclinical safety studies (rodents, pigs, primates) Development of clinical cell injection method Adequate outcome assessment methods for a valid appraisal of patient Guest et al. 2013

56 Miami Project Schwann Cell Team

57 Milestones Dec 07 Organized SC clinical trial team, weekly meetings Developed GMP cell manufacturing procedures for human SCs Tested toxicology and tumorigenicity in rats -SCI studies in pigs to determine safest cell injection procedure More toxicology and tumorigenicity studies on rats -Studies in pigs to determine safest dose and volume for clinical protocol July 11 July 12 Dec 12 Submitted IND application to the FDA Approval! Performed 1 st transplantation

58 IND submission

59 The autologous hsc trial Open label, unblinded, non-randomized and non- placebo dose escalation study, looking at safety of transplantation of autologous hscs with long term follow-up in subjects with subacute SCI Cell preparation protocols are FDA approved and cell processing is conducted in a facility with extensive experience producing clinical grade cells (GMP) Piece of sural nerve is harvested within 5 days post- injury

60 What did the FDA require? Studies to detect: Tumors? Biodistribution? Appropriate dose (MTD)? Toxicity? Cell survival for 6 mo? Characterization of cell product (GMP) Every substance FDA approved Each step in protocol recorded + witnessed Mode of SC injection

61 Challenges $$$, rats, ramping up personnel (training), paper work Documentation of outcome of EVERY rat, including cause of death SOPs for every step in every procedure Sectioning of entire brain and spinal cord to detect migration of transplanted SCs Certified Pathologist to examine sections

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63 Next steps? New clinical trial for combination approach Translatable: -Improve implant survival -Slow release of added components in implant and beyond -Growth factor gradients beyond implant -Provide environment for axon growth/implant and beyond -Provide oxygen for early implant Collaborate with bioengineers

64 Dream biomaterial Non-toxic, biocompatible, biodegradable Non-inflammatory, non scar response Injectable as fluid (with cells) for closed injury Gels upon filling injury site, linear arrangement (tunnels) Promotes axon growth Protects viability of cells Slowly releases growth factors, other

65 Funding: MBB NIH Fellowship NIH research grant (3 Javits Awards) Renewed every 5 or 7 yr Private Foundations Donors to The Miami Project, 25 yr The Buoniconti Foundation The C and D Reeve Foundation, 15 yr Christine E. Lynn Disting Professor, 11 yr State of Florida

66 If you want to go quickly, go alone. If you want to go far, go together. African Proverb

67 Thank You! The Miami Project to Cure Paralysis

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69 Neurotrophins Family Members: -Nerve Growth Factor (NGF) -Brain Derived Neurotrophic Factor (BDNF) -Neurotrophin 3 (NT 3) -Neurotrophin 4 and/or 5 (NT- 4/5) Functions: -Control of proliferation, survival, differentiation -Central and peripheral nervous systems -Developing and adult nervous systems

70 Inclusion Criteria Traumatic thoracic SCI between levels T3-T11 ASIA grade A, years of age Consents to spinal nerve biopsy, implantation, one year follow-up and 5-year protocol Dose Escalation Cohort Target dose Injections Total volume # subjects 1 5 x µl x µl x µl 3

71 Exclusion criteria Penetrating injury Spinal cord transection Lesions involving conus Multi-system trauma Closed head injury Severe neuropathic pain at admission BMI < 35

72 Outcomes 1 outcome assess safety Serious adverse events Changes in sensory and motor scores Changes in pain or spasticity Electrophysiology for MEP and SSEP MRI 2 outcome evaluate functional scales Functional Independence Measure (FIM) Spinal Cord Independence Measure (SCIM) Autonomic (BP, HR, tilt table response, etc.) Follow-up of above for one year Follow-up (MRI annually for 4 years after first year)