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Intraspinal Neural Stem Cell Transplantation in Amyotrophic Lateral Sclerosis: Phase 1 Trial Outcomes

Intraspinal Neural Stem Cell Transplantation in Amyotrophic Lateral Sclerosis: Phase 1 Trial... RESEARCH ARTICLE Intraspinal Neural Stem Cell Transplantation in Amyotrophic Lateral Sclerosis: Phase 1 Trial Outcomes 1 2 1 Eva L. Feldman, MD, PhD, Nicholas M. Boulis, MD, PhD, Junguk Hur, PhD, 3 4 2 Karl Johe, PhD, Seward B. Rutkove, MD, Thais Federici, PhD, 5 5 6 Meraida Polak, RN, Jane Bordeau, RN, Stacey A. Sakowski, PhD, and Jonathan D. Glass, MD Objective: The US Food and Drug Administration–approved trial, “A Phase 1, Open-Label, First-in-Human, Feasibility and Safety Study of Human Spinal Cord-Derived Neural Stem Cell Transplantation for the Treatment of Amyotrophic Lateral Sclerosis, Protocol Number: NS2008-1,” is complete. Our overall objective was to assess the safety and feasi- bility of stem cell transplantation into lumbar and=or cervical spinal cord regions in amyotrophic lateral sclerosis (ALS) subjects. Methods: Preliminary results have been reported on the initial trial cohort of 12 ALS subjects. Here, we describe the safety and functional outcome monitoring results for the final trial cohort, consisting of 6 ALS subjects receiving 5 unilateral cervical intraspinal neural stem cell injections. Three of these subjects previously received 10 total bilateral lumbar injections as part of the earlier trial cohort. All injections utilized a novel spinal-mounted stabilization and injection device to deliver 100,000 neural stem cells per injection, for a dosing range up to 1.5 million cells. Subject assessments included detailed pre- and postsurgical neurological outcome measures. Results: The cervical injection procedure was well tolerated and disease progression did not accelerate in any sub- ject, verifying the safety and feasibility of cervical and dual-targeting approaches. Analyses on outcome data revealed preliminary insight into potential windows of stem cell biological activity and identified clinical assessment measures that closely correlate with ALS Functional Rating Scale-Revised scores, a standard assessment for ALS clinical trials. Interpretation: This is the first report of cervical and dual-targeted intraspinal transplantation of neural stem cells in ALS subjects. This approach is feasible and well-tolerated, supporting future trial phases examining therapeutic dos- ing and efficacy. ANN NEUROL 2014;75:363–373 1–10 here is growing interest in the use of stem cells as others demonstrate that human spinal stem cells (HSSCs) Ta therapy in amyotrophic lateral sclerosis (ALS), a produce protective growth factor profiles, differentiate lethal neurological disorder characterized by the degener- into neurons, form synapses with host motor neurons, ation of motor neurons. Stem cells offer a means to and have beneficial effects after intraspinal transplanta- replace lost cells, provide neurotrophic support, and tion in G93A-SOD1 rats, an established model of 1,7–10 2–6 improve the diseased microenvironment. Preclinical ALS. in vitro and in vivo evidence supports the therapeutic In 2009, the US Food and Drug Administration translation of stem cells, and studies by our group and (FDA) approved a phase 1 clinical trial examining the View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24113 Received Oct 3, 2013, and in revised form Jan 17, 2014. Accepted for publication Jan 25, 2014. Address correspondence to Dr Feldman, University of Michigan, 109 Zina Pitcher Place, 5017 AAT-BSRB, Ann Arbor, MI 48109. E-mail: efeldman@umich.edu 1 2 From the Department of Neurology, University of Michigan, Ann Arbor, MI; Department of Neurosurgery, Emory University School of Medicine, 3 4 Atlanta, GA; Neuralstem, Rockville, MD; Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 5 6 Department of Neurology, Emory University School of Medicine, Atlanta, GA and A. Alfred Taubman Medical Research Institute, University of Michigan, Ann Arbor, MI. Additional supporting information can be found in the online version of this article. V 2014 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. 363 ANNALS of Neurology 7,15,16 design consisting of 5 subject cohorts. Subject selection safety and feasibility of HSSC injections into the spinal criteria, demographics, and inclusion and enrollment criteria for cords of 18 ALS subjects. HSSCs were delivered using a 15,16 Groups A to C have been previously described. For Groups novel intraspinal stabilization and injection device devel- 11–14 D and E, inclusion criteria were the same as for Group B with oped by our group. The first 12 trial subjects, repre- the additional requirement of demonstrable arm weakness with senting Cohorts A to C, received HSSC transplants into an ALS Functional Rating Scale-Revised (ALSFRS-R) arm sub- the L2–L4 lumbar segments of the spinal cord. Group A score between 1 and 3; all Group E subjects were recruited subjects were nonambulatory and received 5 unilateral from Group C and had received prior lumbar intraspinal stem (A1, n 5 3) or 10 total bilateral (A2, n 5 3) lumbar 17 cell injections. Detailed inclusion and exclusion criteria are injections. Subjects in Groups B and C were ambulatory available at http:==www.clinicaltrials.gov=ct2=show=NCT0134 and received 5 unilateral (n 5 3) or 10 total bilateral (n 5 3) lumbar injections, respectively. As previously described, interim results from these first 12 subjects Neural Stem Cell Selection demonstrated no serious adverse events associated with The NSI-566RSC HSSC line used in the trial has been previ- 15,16 6,18,19 ously described. The cells are stored under Current Good HSSC transplantation. Manufacturing Practice (CGMP) conditions and delivered to Those encouraging results along with the critical 15,16 the surgery site at a concentration of 10,000 cells=ll. Cell need to maintain respiratory function in ALS subjects viability was assessed prior to each surgery to ensure the enabled FDA approval to complete HSSC injections into required viability of at least 70% to proceed with C3–C5 cervical segments of the spinal cord, the region 15,16 transplantation. where motor neurons involved in diaphragmatic function reside. To support these injections, the lumbar stabiliza- Cervical Stem Cell Transplantation Approach tion and injection device was adapted and optimized for For Cohorts D and E, adaptations were made to the lumbar 12–14 Ambulatory sub- cervical intraspinal HSSC delivery. 11–16 stabilization and injection device and surgical procedure to jects in Groups D (n 5 3) and E (n 5 3) received 5 accommodate cervical injections, including redesign of the unilateral cervical injections. Based on previous preclini- mounting platform to adhere the device caudally to the C7 ver- 12–14 cal data demonstrating enhanced therapeutic efficacy of tebrae and rostrally at the base of the skull. Briefly, stand- HSSC transplantation when injections were targeted to ard anesthetic and monitoring techniques were adhered to 2,4 15,16 similar to those for lumbar injections, and the surgical pro- multiple spinal cord segments, subjects in Group E cedure for Groups D and E involved a C3–C5 laminectomy. were the same subjects who previously received bilateral Subjects received 5 unilateral injections spaced 4mm apart. Ten lumbar injections as part of Group C. This cohort repre- microliters was delivered at a rate of 5ll=min over 2 minutes, sents the first examination of the feasibility of targeting for a total of 500,000 cells in the 5 injections. Following com- both lumbar and cervical spinal cord segments in ALS pletion of all injections, the dura and tissue incisions were subjects in separate surgeries. closed and postoperative subject care was managed as previously The phase 1 trial consisting of 18 intraspinal trans- 15,16 described. A conservative lifelong, multiagent immunosup- plantation surgeries in 15 ALS subjects was completed in 15,16 pression approach was employed for the phase 1 trial. For May 2013. Here, we present the functional outcome additional details of the cervical microinjection device, surgical data from the 6 subjects undergoing cervical stem cell procedure, and immunosuppression regimen for subjects in transplantation surgery, including 3 subjects receiving Groups D and E, refer to our technical approach and safety both bilateral lumbar and unilateral cervical HSSC trans- outcome report. plants. Data are also presented from the continued follow-up of the first 12 subjects receiving lumbar intra- Subject Assessments All subjects received magnetic resonance imaging (MRI) during spinal HSSC transplants. Overall, results demonstrate screening to calculate precise injection positioning and to serve that HSSCs can be safely transplanted into both lumbar as a baseline for the assessment of postoperative MRI scans, and=or cervical human spinal cord segments, warranting which will be analyzed and reported separately. To determine future trial phases focused on cellular dosing and thera- progression of disease status, subjects regularly underwent peutic efficacy. standard clinical evaluations as well as regular functional assess- ments, including ALSFRS-R, seated forced vital capacity Patients and Methods (FVC), grip strength assessment (GST), hand-held dynamome- Trial Design and Subject Selection try (HHD), electrical impedance myography (EIM), and blad- 15–17 The goals of this phase 1 trial were to assess the safety and tol- der ultrasounds. Group A subjects were not ambulatory; erability of the surgical procedure and the presence of neural these subjects were evaluated once preoperatively and regularly stem cells in the spinal cord, and to examine the use of immu- following transplantation. All remaining subjects in Groups B, nosuppression in ALS subjects, using a risk escalation study C, and D were evaluated monthly for 3 months before surgery 364 Volume 75, No. 3 Feldman et al.: ALS Stem Cell Transplantation TABLE 1. Subject Demographics Group Surgery Subject Surgery Subject Disease Gender Death, mo Details Number Number Age at Duration Postsurgery Surgery, at Surgery, yr yr A1 Nonambulatory 1 1 61.8 5.2 M 30 unilateral 2 2 43.4 12.7 M lumbar 3 3 51.1 2.1 M 13 A2 Nonambulatory 4 4 37.5 2 M bilateral 5 5 66.3 2.2 M 19 lumbar 6 6 55 2.2 M 9 B Ambulatory 7 7 59 1.6 M unilateral 8 8 41.1 5.6 M lumbar 9 9 54.6 1.3 M 11 C/E Ambulatory 10 10 48.9 11.6 M bilateral 16 50.2 13 lumbar 11 11 39.3 1.6 M and 18 40.7 3 unilateral 12 12 65.1 3 M cervical 17 66.3 4.3 D Ambulatory 13 13 50.3 3.1 M20 unilateral 14 14 54.3 1.8 F7 cervical 15 15 35.2 1.7 F F 5 female; M 5 male. Subject demonstrated features of bulbar onset amyotrophic lateral sclerosis. to establish a standard slope of disease progression and regularly with ALSFRS-R scores. Finally, we calculated progression rate following transplantation. Group E subjects previously received slopes for ALSFRS-R scores and GST outcomes based on data lumbar stem cell transplants as Group C; therefore, functional points across 9-month sliding windows to determine whether assessment schedules were already underway prior to surgery there were periods where progression rates were attenuated or and were continued regularly following cervical transplantation. improved relative to the presurgical progression rate. These The schedule of all pre- and postoperative assessments is sum- analyses were performed for Group E subjects (individuals who marized in Supplementary Table 1. received both lumbar and cervical transplantation), as they had Although this was a phase 1 trial and functional outcome the largest amount of available assessment data. Plotted values data were collected for the purpose of assessing safety, secondary represent slopes generated from the available data points within analyses of these data were performed as a means to gain each 9-month window. Best-fit curves were then generated for insight into how cellular transplantation affected disease pro- each subject using fourth-order polynomial analyses. All statisti- gression rates and to inform outcome assessment approaches in cal analyses and curve fitting utilized R version 3.0.1 future trial phases. Presurgical disease progression rates for the (http:==cran.r-project.org=) and Prism 6 for Windows (Graph- various functional outcome measures were first calculated using Pad, SanDiego, CA). linear regression analyses for subjects with multiple available presurgical outcome assessment data points. These slopes were Results utilized to determine whether postsurgical assessment data Subject Selection and General Surgical points at 6, 9, 12, and 15 months were improved relative to Outcomes predicted points extrapolated from the presurgical progression Subject demographics for all cohorts are presented in rates. In addition, Pearson correlation analyses were performed using available data points for the various functional measures Table 1. Enrolled subjects included 13 males and 2 to determine which outcome assessments most closely correlated females ranging in age from 35 to 66 years old. Disease March 2014 365 ANNALS of Neurology FIGURE 1: Neuropathological findings in Subject 14. (A) Gross image of cervical spinal cord at the time of autopsy. Serial sec- tions through the region of transplantation did not demonstrate regions of cystic change, hemorrhage, or significant tissue dis- ruption. (B) Representative cross section showing intact cord morphology using hematoxylin and eosin (H&E) staining. There is a nest of cells (circled) that are not intrinsic to the spinal cord, and do not stain with glial or neuronal markers (not shown). (C) Higher power of circled region in B showing the morphology of these cells, which is reminiscent of the morphology of the stem cells prior to transplantation (inset, H&E). duration ranged between 1.3 and 13 years at the time of formation, or inflammatory reaction within the sites of surgery. All Group E subjects, 1 of 2 trial cohorts desig- transplantation. A representative example of the initial nated to receive cervical stem cell transplants, previously postmortem morphological findings is presented for Sub- received lumbar stem cell transplants as Group C. In ject 14 (Fig 1). This subject received cervical injections 15–17 total, 15 ALS subjects underwent 18 surgeries. and died 7 months postsurgery. There were no morpho- Overall, the procedure was well tolerated across all logical abnormalities within the sites of transplantation; cohorts, with minimal perioperative or postoperative however, a nest of cells likely composed of the trans- complications. Only a nominal number of serious planted cells was identified. adverse events were observed during the course of the phase 1 trial. For cervical injections in Groups D and Functional Outcome Measures E, detailed reports on the intraoperative and the immedi- Subjects regularly (see Supplementary Table 1) under- ate postoperative surgical outcomes and morbidity data went clinical assessments. Interim results from Subjects 1 are presented in our recent technical approach and safety to 12 demonstrated no obvious acceleration of disease outcome report. progression, and Subject 11 in Group C demonstrated At this point, 7 subject deaths have occurred (see modest improvements in postoperative ALSFRS-R, Table 1). As previously reported, Subject 6 died suddenly HHD, and EIM measurements. Continued functional and unexpectedly 8 months postsurgery due to a congen- outcome measure monitoring for Group A and B sub- ital cardiac defect, and Subject 3 died of respiratory fail- jects is presented and discussed in Supplementary Figures ure associated with disease progression 13 months 1 and 2, respectively. Overall, these subjects continued to 15,16 postsurgery. Subjects 1, 5, 9, 13, and 14 also died of demonstrate outcomes consistent with disease progres- respiratory complications associated with ALS disease sion, but no acceleration of the disease course. progression at 30, 19, 11, 20, and 7 months postsurgery, respectively. All patients underwent autopsy for analysis GROUP D: CERVICAL INJECTION. Functional out- of tissue response to implantation and for the identifica- comes are presented in Figure 2. Subjects 13 and 14 tion of the continued presence of the transplanted cells both had features of bulbar ALS. Subject 13 developed within the spinal cord. The detailed results of these anal- cervical kyphosis and died 20 months following trans- yses will be reported separately. Briefly, standard patho- plantation, and Subject 14 died 200 days following trans- logical analysis showed no evidence of hemorrhage, cyst plantation. Although other clinical markers remained 366 Volume 75, No. 3 Feldman et al.: ALS Stem Cell Transplantation FIGURE 2: Evaluation of disease progression in Group D subjects. Disease progression for Subjects 13 to 15 as measured by (A) Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R), (B) forced vital capacity (FVC), (C, D) hand-held dynamometry (HHD), (E, F) grip strength assessment (GST), and (G, H) electrical impedance myography (EIM). HHD is shown as a composite megascore for upper (C) or lower (D) extremities, normalized to the percentage of the score at baseline. GST data are presented for left (E) and right (F) sides. EIM is shown as 50kHz phase all muscle average for upper (G) or lower (H) extremity muscles. X-axis represents days pre- or postsurgery (day of surgery 5 day 0). Note that there were no precipitous declines in function after surgery for any subject. Note that a score of 0 for Subject 14 indicates subject death on the day post-transplantation indicated on the x-axis. stable, Subject 15 demonstrated a modest decline in progressive form of ALS. Subjects 11 and 12 had ALSFRS-R and HHD following transplantation, reflect- improved ALSFRS-R scores, steady FVC values, and ing a progression that appeared slower than what is typi- modest declines in HHD megascores following HSSC cally expected for ALS. transplantation, suggesting some progression of disease accompanied by multiple improved functional measures. GROUP E: DUAL-TARGETED INJECTIONS. Func- tional outcome measures are presented in Figure 3. Sub- ADVANCED ANALYSES OF FUNCTIONAL OUTCOME ject 10 had a long disease duration and maintained a MEASURES. We performed additional analyses to gain steady ALSFRS-R score accompanied by mild declines in insight into the effects of the intervention on disease pro- other functional measures, suggestive of a very slowly gression and to identify appropriate functional outcome March 2014 367 ANNALS of Neurology FIGURE 3: Evaluation of disease progression in Group E subjects. Disease progression for Subjects 10 to 12 as measured by (A) Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R), (B) forced vital capacity (FVC), (C, D) hand-held dynamometry (HHD), (E, F) grip strength assessment (GST), and (G, H) electrical impedance myography (EIM). HHD is shown as a composite megascore for lower (C) or upper (D) extremities, normalized to the percentage of the score at baseline. GST data are presented for left (E) and right (F) sides. EIM is shown as 50kHz phase all muscle average for upper (G) or lower (H) extremity muscles. X-axis represents days before or after lumbar surgery (day of surgery 5 day 0). Note that there were no precipitous declines in function after surgery for any subject. Note that Group E subjects are subjects initially enrolled in Group C who received lumbar stem cell injections, and the short dotted vertical bars indicate the number of days after the first sur- gery when the second stem cell transplantations (cervical injections) were administered. Note that Subject 11 (purple lines) showed apparent improvement in ALSFRS-R and upper and lower extremity HHD. measures for future trial phases. Comparison of postsurgi- coordinated most closely with ALSFRS-R scores, Pearson cal outcome data to predicted outcome points extrapolated correlations were calculated between data points for the from presurgical disease progression slopes revealed various functional outcome measures. Results indicate improvements in a significant number of measures at 6, 9, that GST measures most closely reflect ALSFRS-R values 12, and 15 months postsurgery (Table 2). Of the 8 out- throughout the study period (Fig 4), suggesting that come assessments, at least 5 measures were improved in ALSFRS-R and GST assessments will provide important >50% of subjects at each time point relative to the pre- outcome information in future trial phases. dicted outcome values extrapolated from presurgical pro- As shown in Figure 5, analysis of ALSFRS-R scores gression rates. To identify which functional assessments for Group E subjects exhibits improved outcomes (slope 368 Volume 75, No. 3 Feldman et al.: ALS Stem Cell Transplantation March 2014 369 TABLE 2. Functional Outcomes versus Predicted Outcomes Based on Presurgical Assessments Time Postsurgery 6 Months 9 Months 12 Months 15 Months Assessment Subjects Subjects Ratio Subjects Subjects Ratio Subjects Subjects Ratio Subjects Subjects Ratio Measure with with with with with with with with Presurgical Improved Presurgical Improved Presurgical Improved Presurgical Improved Slope Outcome, Slope Outcome, Slope Outcome, Slope Outcome, a a a a Data, No. Data, No. No. Data, No. Data, No. No. No. No. ALSFRS 9 3 33% 9 4 44% 9 3 33% 9 3 33% FVC seated 9 3 33% 9 3 33% 9 3 33% 9 3 33% HHD upper 9 7 78%98 89%98 89%97 78% HHD lower 9 5 56% 9 4 44% 9 4 44% 9 5 56% GST left 8 7 88%88 100%87 88%87 88% GST right 8 6 75%86 75%86 75%86 75% EIM upper 4 3 75%43 75%43 75%43 75% EIM lower 10 6 60% 10 6 60% 10 6 60% 10 7 70% Slopes were utilized to determine whether postsurgical assessment data points at 6, 9, 12, and 15 months were improved relative to predicted outcomes extrapolated from the presurgi- cal progression rate slopes ALSFRS 5 Amyotrophic Lateral Sclerosis Functional Rating Scale; EIM 5 electrical impedance myography; FVC 5 forced vital capacity; GST 5 grip strength assessment; HHD 5 hand-held dynamometry. Ratios indicated in bold represent outcome measures that are improved in over 50% of subjects relative to points predicted from presurgical slopes. ANNALS of Neurology FIGURE 4: Pearson correlations for outcome assessment measures. ALSFRS 5 Amyotrophic Lateral Sclerosis Functional Rating Scale; EIM 5 electrical impedance myography; FVC 5 forced vital capacity; GST 5 grip strength assessment; HHD 5 hand-held dynamometry. 15,16 values higher than the presurgical slope at baseline reflect results from Groups A to C, representing 12 subjects improved or attenuated progression rates during the des- who received lumbar injections, supported the completion ignated window) beginning within the first month post- of the final trial with Cohorts D and E, examining cervical surgery, with slopes remaining positive for windows injections in 6 ALS subjects. Notably, the final 3 subjects beginning up to 6 months postsurgery. Although the rate receiving cervical injections previously received bilateral of benefit then decreases over time, the overall progres- lumbar injections. Our study represents the first report of sion rate generally remains attenuated relative to the pre- successful intraspinal stem cell transplantation into the cer- surgical slope through the time of the second surgery. vical spinal cord and of successful repeated intraspinal stem Positive slopes are again observed across treatment win- cell transplantation into lumbar and cervical spinal cord dows beginning at approximately 13 to 14 months post- segments in ALS subjects in an FDA-approved trial. Our surgery for these subjects, reflecting the second HSSC ability to directly inject stem cells to target motor neurons transplant in Subjects 10, 11, and 12 at 490, 532, and in the region of the cervical spinal cord responsible for res- 464 days, respectively. This bimodal representation of piration represents a significant advance in the field of cel- HSSC benefit suggests that the biological activity of the lular therapy. In parallel, the dual-targeting approach, that cells shows the greatest benefits in the 6 months immedi- is, both cervical and lumbar transplantation, has the poten- ately following the surgeries (see Fig 5B), continuing to tial to preserve respiratory function and improve motor provide some benefit throughout the study evaluation function in ALS subjects. What is now required are future period. Similar analyses of GST data for this cohort studies to determine whether these approaches provide sus- reflect comparable trends (data not shown). tained clinical improvement in ALS. Of the 15 subjects in the phase 1 trial, 6 subjects Discussion died of their disease and 1 subject died of a congenital In this completed FDA-approved phase 1 trial, 18 intraspi- heart defect unrelated to ALS between 7 and 30 months nal transplantation surgeries in 15 ALS subjects were per- after surgery. Of the 8 subjects who are still alive, 3 of formed following a risk escalation paradigm, progressing them (Subjects 2, 8, and 10) had a long disease course from nonambulatory to ambulatory subjects, lumbar to prior to surgery (5.6, 11.6, and 12.7 years of known dis- cervical spinal cord segments, and unilateral to bilateral ease), likely representing atypical ALS, and have had little injections across 5 cohorts. The encouraging interim change in the trajectory of their disease. Subjects 7, 11, 370 Volume 75, No. 3 Feldman et al.: ALS Stem Cell Transplantation FIGURE 5: Preliminary analysis of potential windows of human spinal stem cell (HSSC) biological activity in Subjects 10 to 12. To identify the most biologically active period of the injected HSSCs, postsurgery data points for Group E subjects were divided into a series of 9-month windows, beginning each month postsurgery, and slopes were calculated across each window. Slopes were also calculated using Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) data points for the presurgical window. (A) The top panel demonstrates ALSFRS-R scores for Group E subjects during the presurgical period (green) and representative ranges associated with the various sliding postsurgical 9-month windows (dark blue). The bottom panel demonstrates the slopes obtained for each sliding window, with the x-axis corresponding to the first month for each 9- month window (ie, window 1 corresponds to months 1–10 postsurgery, window 2 corresponds to months 2–11 postsurgery, window 3 corresponds to months 3–12 postsurgery, etc). The first plotted slope for each subject corresponds to their presurgi- cal progression rate. Slope values higher than the presurgical slope at baseline represent improved or attenuated progression rates during the designated window. Note that the starting month of the final sliding window for each patient coincides with the dates of the second surgery, which occur at 17.5, 19, and 16.6 months after the initial Cohort C surgery (time 0) for Sub- jects 10, 11, and 12, respectively. (B) The presurgical slope and postsurgical slopes associated with the window correlating to the peak benefit windows for both the lumbar and cervical postsurgery time frames are summarized. 12, and 15, who are alive with very slowly progressive or slopes to post-transplant data revealed that >50% of sub- stabilized disease, had 2 clinical characteristics in com- jects demonstrated improvement across multiple clinical mon: these individuals had no bulbar features of ALS, measures at 6, 9, 12, and 15 months postsurgery. Look- and surgical transplantation occurred early within the ing specifically at ALSFRS-R scores at the 9-month time course of their disease (average of 2 years and 1 month point, the subjects who demonstrated improvements were after symptom onset at the time of surgery). These pre- part of Groups B, C, and E and exhibited an average dis- liminary results raise the possibility that intraspinal stem ease duration <2 years prior to surgery, again suggesting cell transplantation of ALS subjects with no bulbar symp- that only subjects early in the disease course may experi- toms early in the course of their disease could slow dis- ence clinical benefit. However, our experience did dem- ease progression or even allow for functional onstrate a wide variation in presurgical progression rates improvement. for those individuals with multiple data points, empha- The majority of ALS trials utilize subject survival sizing the importance of sufficient lead-in data to deter- and ALSFRS-R scores for primary outcome measures. mine efficacy. Average declines of 21.1 ALSFRS-R score Our data demonstrate that GST most closely correlates per month (213.32 per year) have been reported ; how- with the ALSFRS-R scores. Comparisons of presurgical ever, the varied slopes we observed and the heterogeneous March 2014 371 ANNALS of Neurology E.L.F., N.M.B., S.B.R.), the ALS Association (E.L.F.), presentation of ALS emphasize the need for subject- and Neuralstem. Additional support for tissue and data specific baseline data. We acknowledge that this study was not powered analysis was provided by the NIH National Institute on Aging (5P50AG025688; J.D.G.) and the A. Alfred Taub- to determine efficacy and there was no control arm. In man Medical Research Institute (E.L.F., N.M.B.). addition, some subjects exhibited a significant disease burden prior to surgery and were unlikely to show bene- We thank the study participants and their families for their trust and dedication to advancing the field of ALS fit, sufficient preclinical data points were unavailable for therapeutics; the trial Data Safety Monitoring Board, some subjects, and best-fit presurgical slopes were not always significantly powered given the number of avail- chaired by Dr Z. Simmons; Dr T. G. Hazel for provid- able data points. Despite these limitations, we were able ing the CGMP stem cells; L. Shaw for help with subject assessments; Dr M. Gearing for assistance with neuropa- to identify potential possible therapeutic windows in our thology; the staff of the Emory ALS Center; and J. Duell advanced evaluation of Group C=E outcome data. Of note, the 3 subjects in this cohort received the highest for assistance with table preparation. number of injections and demonstrated the largest effects Authorship on progression rates, suggesting that more injections are better, consistent with the neuroprotective mechanism of E.L.F., N.M.B., and J.D.G. designed the study. K.J. pro- 7,9 action hypothesized for this cell therapy. The ability to vided critical study design input. N.M.B., T.F., M.P., successfully administer 1.5 million HSSCs to ALS sub- J.B., and J.D.G. contributed to data acquisition. E.L.F., jects over 15 total injections in Group E subjects into N.M.B., J.H., S.B.R., S.A.S., and J.D.G. were responsi- both lumbar and cervical spinal cord segments over the ble for data analysis and interpretation. E.L.F. and S.A.S. course of 2 surgeries is an important first step in evaluat- drafted the manuscript. All authors critically edited the ing the tolerance of the spinal cord for multiple HSSC content of the article and approved the final version. transplantation procedures. The observed bimodal distri- Potential Conflicts of Interest bution in the 9-month sliding window slope analysis sug- gests there are maximal periods of benefit that correlate N.M.B.: consultancy, Neuralstem; share of sale of Cleve- with the 2 surgical interventions. Furthermore, as the land Clinic Foundation subsidiary IntElect to Boston Sci- bell-shaped benefit curve associated with each interven- entific; patents, licensed to Neuralstem. K.J.: stock, tion is likely due to disease progression, increasing the Neuralstem; patents, assigned to Neuralstem. S.B.R.: total cell dose and applying multiple applications of the equity, consultancy, scientific advisor, Board of Directors, stem cells may increase both the length and magnitude Skulpt; patents, assigned to Skulpt. of potential benefit. 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Platform and cannula design spinal cord stem cell line, NSI-566RSC, and its induction to func- improvements for spinal cord therapeutics delivery. Neurosurgery tional motoneurons. J Tissue Eng Regen Med 2010;4:181–193. 2011;69(2 suppl):ons147–ons154; discussion ons155. 19. Johe KK, Hazel TG, Muller T, et al. Single factors direct the differ- 13. Raore B, Federici T, Taub J, et al. Cervical multilevel intraspinal entiation of stem cells from the fetal and adult central nervous stem cell therapy: assessment of surgical risks in Gottingen mini- system. Genes Dev 1996;10:3129–3140. pigs. Spine (Phila Pa 1976) 2011;36:E164–E171. 14. Riley J, Federici T, Park J, et al. Cervical spinal cord therapeutics 20. Healy BC, Schoenfeld D. Comparison of analysis approaches for delivery: preclinical safety validation of a stabilized microinjection phase III clinical trials in amyotrophic lateral sclerosis. Muscle platform. Neurosurgery 2009;65:754–761; discussion 761–762. Nerve 2012;46:506–511. March 2014 373 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Neurology Pubmed Central

Intraspinal Neural Stem Cell Transplantation in Amyotrophic Lateral Sclerosis: Phase 1 Trial Outcomes

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© 2014 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association
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10.1002/ana.24113
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Abstract

RESEARCH ARTICLE Intraspinal Neural Stem Cell Transplantation in Amyotrophic Lateral Sclerosis: Phase 1 Trial Outcomes 1 2 1 Eva L. Feldman, MD, PhD, Nicholas M. Boulis, MD, PhD, Junguk Hur, PhD, 3 4 2 Karl Johe, PhD, Seward B. Rutkove, MD, Thais Federici, PhD, 5 5 6 Meraida Polak, RN, Jane Bordeau, RN, Stacey A. Sakowski, PhD, and Jonathan D. Glass, MD Objective: The US Food and Drug Administration–approved trial, “A Phase 1, Open-Label, First-in-Human, Feasibility and Safety Study of Human Spinal Cord-Derived Neural Stem Cell Transplantation for the Treatment of Amyotrophic Lateral Sclerosis, Protocol Number: NS2008-1,” is complete. Our overall objective was to assess the safety and feasi- bility of stem cell transplantation into lumbar and=or cervical spinal cord regions in amyotrophic lateral sclerosis (ALS) subjects. Methods: Preliminary results have been reported on the initial trial cohort of 12 ALS subjects. Here, we describe the safety and functional outcome monitoring results for the final trial cohort, consisting of 6 ALS subjects receiving 5 unilateral cervical intraspinal neural stem cell injections. Three of these subjects previously received 10 total bilateral lumbar injections as part of the earlier trial cohort. All injections utilized a novel spinal-mounted stabilization and injection device to deliver 100,000 neural stem cells per injection, for a dosing range up to 1.5 million cells. Subject assessments included detailed pre- and postsurgical neurological outcome measures. Results: The cervical injection procedure was well tolerated and disease progression did not accelerate in any sub- ject, verifying the safety and feasibility of cervical and dual-targeting approaches. Analyses on outcome data revealed preliminary insight into potential windows of stem cell biological activity and identified clinical assessment measures that closely correlate with ALS Functional Rating Scale-Revised scores, a standard assessment for ALS clinical trials. Interpretation: This is the first report of cervical and dual-targeted intraspinal transplantation of neural stem cells in ALS subjects. This approach is feasible and well-tolerated, supporting future trial phases examining therapeutic dos- ing and efficacy. ANN NEUROL 2014;75:363–373 1–10 here is growing interest in the use of stem cells as others demonstrate that human spinal stem cells (HSSCs) Ta therapy in amyotrophic lateral sclerosis (ALS), a produce protective growth factor profiles, differentiate lethal neurological disorder characterized by the degener- into neurons, form synapses with host motor neurons, ation of motor neurons. Stem cells offer a means to and have beneficial effects after intraspinal transplanta- replace lost cells, provide neurotrophic support, and tion in G93A-SOD1 rats, an established model of 1,7–10 2–6 improve the diseased microenvironment. Preclinical ALS. in vitro and in vivo evidence supports the therapeutic In 2009, the US Food and Drug Administration translation of stem cells, and studies by our group and (FDA) approved a phase 1 clinical trial examining the View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24113 Received Oct 3, 2013, and in revised form Jan 17, 2014. Accepted for publication Jan 25, 2014. Address correspondence to Dr Feldman, University of Michigan, 109 Zina Pitcher Place, 5017 AAT-BSRB, Ann Arbor, MI 48109. E-mail: efeldman@umich.edu 1 2 From the Department of Neurology, University of Michigan, Ann Arbor, MI; Department of Neurosurgery, Emory University School of Medicine, 3 4 Atlanta, GA; Neuralstem, Rockville, MD; Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 5 6 Department of Neurology, Emory University School of Medicine, Atlanta, GA and A. Alfred Taubman Medical Research Institute, University of Michigan, Ann Arbor, MI. Additional supporting information can be found in the online version of this article. V 2014 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. 363 ANNALS of Neurology 7,15,16 design consisting of 5 subject cohorts. Subject selection safety and feasibility of HSSC injections into the spinal criteria, demographics, and inclusion and enrollment criteria for cords of 18 ALS subjects. HSSCs were delivered using a 15,16 Groups A to C have been previously described. For Groups novel intraspinal stabilization and injection device devel- 11–14 D and E, inclusion criteria were the same as for Group B with oped by our group. The first 12 trial subjects, repre- the additional requirement of demonstrable arm weakness with senting Cohorts A to C, received HSSC transplants into an ALS Functional Rating Scale-Revised (ALSFRS-R) arm sub- the L2–L4 lumbar segments of the spinal cord. Group A score between 1 and 3; all Group E subjects were recruited subjects were nonambulatory and received 5 unilateral from Group C and had received prior lumbar intraspinal stem (A1, n 5 3) or 10 total bilateral (A2, n 5 3) lumbar 17 cell injections. Detailed inclusion and exclusion criteria are injections. Subjects in Groups B and C were ambulatory available at http:==www.clinicaltrials.gov=ct2=show=NCT0134 and received 5 unilateral (n 5 3) or 10 total bilateral (n 5 3) lumbar injections, respectively. As previously described, interim results from these first 12 subjects Neural Stem Cell Selection demonstrated no serious adverse events associated with The NSI-566RSC HSSC line used in the trial has been previ- 15,16 6,18,19 ously described. The cells are stored under Current Good HSSC transplantation. Manufacturing Practice (CGMP) conditions and delivered to Those encouraging results along with the critical 15,16 the surgery site at a concentration of 10,000 cells=ll. Cell need to maintain respiratory function in ALS subjects viability was assessed prior to each surgery to ensure the enabled FDA approval to complete HSSC injections into required viability of at least 70% to proceed with C3–C5 cervical segments of the spinal cord, the region 15,16 transplantation. where motor neurons involved in diaphragmatic function reside. To support these injections, the lumbar stabiliza- Cervical Stem Cell Transplantation Approach tion and injection device was adapted and optimized for For Cohorts D and E, adaptations were made to the lumbar 12–14 Ambulatory sub- cervical intraspinal HSSC delivery. 11–16 stabilization and injection device and surgical procedure to jects in Groups D (n 5 3) and E (n 5 3) received 5 accommodate cervical injections, including redesign of the unilateral cervical injections. Based on previous preclini- mounting platform to adhere the device caudally to the C7 ver- 12–14 cal data demonstrating enhanced therapeutic efficacy of tebrae and rostrally at the base of the skull. Briefly, stand- HSSC transplantation when injections were targeted to ard anesthetic and monitoring techniques were adhered to 2,4 15,16 similar to those for lumbar injections, and the surgical pro- multiple spinal cord segments, subjects in Group E cedure for Groups D and E involved a C3–C5 laminectomy. were the same subjects who previously received bilateral Subjects received 5 unilateral injections spaced 4mm apart. Ten lumbar injections as part of Group C. This cohort repre- microliters was delivered at a rate of 5ll=min over 2 minutes, sents the first examination of the feasibility of targeting for a total of 500,000 cells in the 5 injections. Following com- both lumbar and cervical spinal cord segments in ALS pletion of all injections, the dura and tissue incisions were subjects in separate surgeries. closed and postoperative subject care was managed as previously The phase 1 trial consisting of 18 intraspinal trans- 15,16 described. A conservative lifelong, multiagent immunosup- plantation surgeries in 15 ALS subjects was completed in 15,16 pression approach was employed for the phase 1 trial. For May 2013. Here, we present the functional outcome additional details of the cervical microinjection device, surgical data from the 6 subjects undergoing cervical stem cell procedure, and immunosuppression regimen for subjects in transplantation surgery, including 3 subjects receiving Groups D and E, refer to our technical approach and safety both bilateral lumbar and unilateral cervical HSSC trans- outcome report. plants. Data are also presented from the continued follow-up of the first 12 subjects receiving lumbar intra- Subject Assessments All subjects received magnetic resonance imaging (MRI) during spinal HSSC transplants. Overall, results demonstrate screening to calculate precise injection positioning and to serve that HSSCs can be safely transplanted into both lumbar as a baseline for the assessment of postoperative MRI scans, and=or cervical human spinal cord segments, warranting which will be analyzed and reported separately. To determine future trial phases focused on cellular dosing and thera- progression of disease status, subjects regularly underwent peutic efficacy. standard clinical evaluations as well as regular functional assess- ments, including ALSFRS-R, seated forced vital capacity Patients and Methods (FVC), grip strength assessment (GST), hand-held dynamome- Trial Design and Subject Selection try (HHD), electrical impedance myography (EIM), and blad- 15–17 The goals of this phase 1 trial were to assess the safety and tol- der ultrasounds. Group A subjects were not ambulatory; erability of the surgical procedure and the presence of neural these subjects were evaluated once preoperatively and regularly stem cells in the spinal cord, and to examine the use of immu- following transplantation. All remaining subjects in Groups B, nosuppression in ALS subjects, using a risk escalation study C, and D were evaluated monthly for 3 months before surgery 364 Volume 75, No. 3 Feldman et al.: ALS Stem Cell Transplantation TABLE 1. Subject Demographics Group Surgery Subject Surgery Subject Disease Gender Death, mo Details Number Number Age at Duration Postsurgery Surgery, at Surgery, yr yr A1 Nonambulatory 1 1 61.8 5.2 M 30 unilateral 2 2 43.4 12.7 M lumbar 3 3 51.1 2.1 M 13 A2 Nonambulatory 4 4 37.5 2 M bilateral 5 5 66.3 2.2 M 19 lumbar 6 6 55 2.2 M 9 B Ambulatory 7 7 59 1.6 M unilateral 8 8 41.1 5.6 M lumbar 9 9 54.6 1.3 M 11 C/E Ambulatory 10 10 48.9 11.6 M bilateral 16 50.2 13 lumbar 11 11 39.3 1.6 M and 18 40.7 3 unilateral 12 12 65.1 3 M cervical 17 66.3 4.3 D Ambulatory 13 13 50.3 3.1 M20 unilateral 14 14 54.3 1.8 F7 cervical 15 15 35.2 1.7 F F 5 female; M 5 male. Subject demonstrated features of bulbar onset amyotrophic lateral sclerosis. to establish a standard slope of disease progression and regularly with ALSFRS-R scores. Finally, we calculated progression rate following transplantation. Group E subjects previously received slopes for ALSFRS-R scores and GST outcomes based on data lumbar stem cell transplants as Group C; therefore, functional points across 9-month sliding windows to determine whether assessment schedules were already underway prior to surgery there were periods where progression rates were attenuated or and were continued regularly following cervical transplantation. improved relative to the presurgical progression rate. These The schedule of all pre- and postoperative assessments is sum- analyses were performed for Group E subjects (individuals who marized in Supplementary Table 1. received both lumbar and cervical transplantation), as they had Although this was a phase 1 trial and functional outcome the largest amount of available assessment data. Plotted values data were collected for the purpose of assessing safety, secondary represent slopes generated from the available data points within analyses of these data were performed as a means to gain each 9-month window. Best-fit curves were then generated for insight into how cellular transplantation affected disease pro- each subject using fourth-order polynomial analyses. All statisti- gression rates and to inform outcome assessment approaches in cal analyses and curve fitting utilized R version 3.0.1 future trial phases. Presurgical disease progression rates for the (http:==cran.r-project.org=) and Prism 6 for Windows (Graph- various functional outcome measures were first calculated using Pad, SanDiego, CA). linear regression analyses for subjects with multiple available presurgical outcome assessment data points. These slopes were Results utilized to determine whether postsurgical assessment data Subject Selection and General Surgical points at 6, 9, 12, and 15 months were improved relative to Outcomes predicted points extrapolated from the presurgical progression Subject demographics for all cohorts are presented in rates. In addition, Pearson correlation analyses were performed using available data points for the various functional measures Table 1. Enrolled subjects included 13 males and 2 to determine which outcome assessments most closely correlated females ranging in age from 35 to 66 years old. Disease March 2014 365 ANNALS of Neurology FIGURE 1: Neuropathological findings in Subject 14. (A) Gross image of cervical spinal cord at the time of autopsy. Serial sec- tions through the region of transplantation did not demonstrate regions of cystic change, hemorrhage, or significant tissue dis- ruption. (B) Representative cross section showing intact cord morphology using hematoxylin and eosin (H&E) staining. There is a nest of cells (circled) that are not intrinsic to the spinal cord, and do not stain with glial or neuronal markers (not shown). (C) Higher power of circled region in B showing the morphology of these cells, which is reminiscent of the morphology of the stem cells prior to transplantation (inset, H&E). duration ranged between 1.3 and 13 years at the time of formation, or inflammatory reaction within the sites of surgery. All Group E subjects, 1 of 2 trial cohorts desig- transplantation. A representative example of the initial nated to receive cervical stem cell transplants, previously postmortem morphological findings is presented for Sub- received lumbar stem cell transplants as Group C. In ject 14 (Fig 1). This subject received cervical injections 15–17 total, 15 ALS subjects underwent 18 surgeries. and died 7 months postsurgery. There were no morpho- Overall, the procedure was well tolerated across all logical abnormalities within the sites of transplantation; cohorts, with minimal perioperative or postoperative however, a nest of cells likely composed of the trans- complications. Only a nominal number of serious planted cells was identified. adverse events were observed during the course of the phase 1 trial. For cervical injections in Groups D and Functional Outcome Measures E, detailed reports on the intraoperative and the immedi- Subjects regularly (see Supplementary Table 1) under- ate postoperative surgical outcomes and morbidity data went clinical assessments. Interim results from Subjects 1 are presented in our recent technical approach and safety to 12 demonstrated no obvious acceleration of disease outcome report. progression, and Subject 11 in Group C demonstrated At this point, 7 subject deaths have occurred (see modest improvements in postoperative ALSFRS-R, Table 1). As previously reported, Subject 6 died suddenly HHD, and EIM measurements. Continued functional and unexpectedly 8 months postsurgery due to a congen- outcome measure monitoring for Group A and B sub- ital cardiac defect, and Subject 3 died of respiratory fail- jects is presented and discussed in Supplementary Figures ure associated with disease progression 13 months 1 and 2, respectively. Overall, these subjects continued to 15,16 postsurgery. Subjects 1, 5, 9, 13, and 14 also died of demonstrate outcomes consistent with disease progres- respiratory complications associated with ALS disease sion, but no acceleration of the disease course. progression at 30, 19, 11, 20, and 7 months postsurgery, respectively. All patients underwent autopsy for analysis GROUP D: CERVICAL INJECTION. Functional out- of tissue response to implantation and for the identifica- comes are presented in Figure 2. Subjects 13 and 14 tion of the continued presence of the transplanted cells both had features of bulbar ALS. Subject 13 developed within the spinal cord. The detailed results of these anal- cervical kyphosis and died 20 months following trans- yses will be reported separately. Briefly, standard patho- plantation, and Subject 14 died 200 days following trans- logical analysis showed no evidence of hemorrhage, cyst plantation. Although other clinical markers remained 366 Volume 75, No. 3 Feldman et al.: ALS Stem Cell Transplantation FIGURE 2: Evaluation of disease progression in Group D subjects. Disease progression for Subjects 13 to 15 as measured by (A) Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R), (B) forced vital capacity (FVC), (C, D) hand-held dynamometry (HHD), (E, F) grip strength assessment (GST), and (G, H) electrical impedance myography (EIM). HHD is shown as a composite megascore for upper (C) or lower (D) extremities, normalized to the percentage of the score at baseline. GST data are presented for left (E) and right (F) sides. EIM is shown as 50kHz phase all muscle average for upper (G) or lower (H) extremity muscles. X-axis represents days pre- or postsurgery (day of surgery 5 day 0). Note that there were no precipitous declines in function after surgery for any subject. Note that a score of 0 for Subject 14 indicates subject death on the day post-transplantation indicated on the x-axis. stable, Subject 15 demonstrated a modest decline in progressive form of ALS. Subjects 11 and 12 had ALSFRS-R and HHD following transplantation, reflect- improved ALSFRS-R scores, steady FVC values, and ing a progression that appeared slower than what is typi- modest declines in HHD megascores following HSSC cally expected for ALS. transplantation, suggesting some progression of disease accompanied by multiple improved functional measures. GROUP E: DUAL-TARGETED INJECTIONS. Func- tional outcome measures are presented in Figure 3. Sub- ADVANCED ANALYSES OF FUNCTIONAL OUTCOME ject 10 had a long disease duration and maintained a MEASURES. We performed additional analyses to gain steady ALSFRS-R score accompanied by mild declines in insight into the effects of the intervention on disease pro- other functional measures, suggestive of a very slowly gression and to identify appropriate functional outcome March 2014 367 ANNALS of Neurology FIGURE 3: Evaluation of disease progression in Group E subjects. Disease progression for Subjects 10 to 12 as measured by (A) Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R), (B) forced vital capacity (FVC), (C, D) hand-held dynamometry (HHD), (E, F) grip strength assessment (GST), and (G, H) electrical impedance myography (EIM). HHD is shown as a composite megascore for lower (C) or upper (D) extremities, normalized to the percentage of the score at baseline. GST data are presented for left (E) and right (F) sides. EIM is shown as 50kHz phase all muscle average for upper (G) or lower (H) extremity muscles. X-axis represents days before or after lumbar surgery (day of surgery 5 day 0). Note that there were no precipitous declines in function after surgery for any subject. Note that Group E subjects are subjects initially enrolled in Group C who received lumbar stem cell injections, and the short dotted vertical bars indicate the number of days after the first sur- gery when the second stem cell transplantations (cervical injections) were administered. Note that Subject 11 (purple lines) showed apparent improvement in ALSFRS-R and upper and lower extremity HHD. measures for future trial phases. Comparison of postsurgi- coordinated most closely with ALSFRS-R scores, Pearson cal outcome data to predicted outcome points extrapolated correlations were calculated between data points for the from presurgical disease progression slopes revealed various functional outcome measures. Results indicate improvements in a significant number of measures at 6, 9, that GST measures most closely reflect ALSFRS-R values 12, and 15 months postsurgery (Table 2). Of the 8 out- throughout the study period (Fig 4), suggesting that come assessments, at least 5 measures were improved in ALSFRS-R and GST assessments will provide important >50% of subjects at each time point relative to the pre- outcome information in future trial phases. dicted outcome values extrapolated from presurgical pro- As shown in Figure 5, analysis of ALSFRS-R scores gression rates. To identify which functional assessments for Group E subjects exhibits improved outcomes (slope 368 Volume 75, No. 3 Feldman et al.: ALS Stem Cell Transplantation March 2014 369 TABLE 2. Functional Outcomes versus Predicted Outcomes Based on Presurgical Assessments Time Postsurgery 6 Months 9 Months 12 Months 15 Months Assessment Subjects Subjects Ratio Subjects Subjects Ratio Subjects Subjects Ratio Subjects Subjects Ratio Measure with with with with with with with with Presurgical Improved Presurgical Improved Presurgical Improved Presurgical Improved Slope Outcome, Slope Outcome, Slope Outcome, Slope Outcome, a a a a Data, No. Data, No. No. Data, No. Data, No. No. No. No. ALSFRS 9 3 33% 9 4 44% 9 3 33% 9 3 33% FVC seated 9 3 33% 9 3 33% 9 3 33% 9 3 33% HHD upper 9 7 78%98 89%98 89%97 78% HHD lower 9 5 56% 9 4 44% 9 4 44% 9 5 56% GST left 8 7 88%88 100%87 88%87 88% GST right 8 6 75%86 75%86 75%86 75% EIM upper 4 3 75%43 75%43 75%43 75% EIM lower 10 6 60% 10 6 60% 10 6 60% 10 7 70% Slopes were utilized to determine whether postsurgical assessment data points at 6, 9, 12, and 15 months were improved relative to predicted outcomes extrapolated from the presurgi- cal progression rate slopes ALSFRS 5 Amyotrophic Lateral Sclerosis Functional Rating Scale; EIM 5 electrical impedance myography; FVC 5 forced vital capacity; GST 5 grip strength assessment; HHD 5 hand-held dynamometry. Ratios indicated in bold represent outcome measures that are improved in over 50% of subjects relative to points predicted from presurgical slopes. ANNALS of Neurology FIGURE 4: Pearson correlations for outcome assessment measures. ALSFRS 5 Amyotrophic Lateral Sclerosis Functional Rating Scale; EIM 5 electrical impedance myography; FVC 5 forced vital capacity; GST 5 grip strength assessment; HHD 5 hand-held dynamometry. 15,16 values higher than the presurgical slope at baseline reflect results from Groups A to C, representing 12 subjects improved or attenuated progression rates during the des- who received lumbar injections, supported the completion ignated window) beginning within the first month post- of the final trial with Cohorts D and E, examining cervical surgery, with slopes remaining positive for windows injections in 6 ALS subjects. Notably, the final 3 subjects beginning up to 6 months postsurgery. Although the rate receiving cervical injections previously received bilateral of benefit then decreases over time, the overall progres- lumbar injections. Our study represents the first report of sion rate generally remains attenuated relative to the pre- successful intraspinal stem cell transplantation into the cer- surgical slope through the time of the second surgery. vical spinal cord and of successful repeated intraspinal stem Positive slopes are again observed across treatment win- cell transplantation into lumbar and cervical spinal cord dows beginning at approximately 13 to 14 months post- segments in ALS subjects in an FDA-approved trial. Our surgery for these subjects, reflecting the second HSSC ability to directly inject stem cells to target motor neurons transplant in Subjects 10, 11, and 12 at 490, 532, and in the region of the cervical spinal cord responsible for res- 464 days, respectively. This bimodal representation of piration represents a significant advance in the field of cel- HSSC benefit suggests that the biological activity of the lular therapy. In parallel, the dual-targeting approach, that cells shows the greatest benefits in the 6 months immedi- is, both cervical and lumbar transplantation, has the poten- ately following the surgeries (see Fig 5B), continuing to tial to preserve respiratory function and improve motor provide some benefit throughout the study evaluation function in ALS subjects. What is now required are future period. Similar analyses of GST data for this cohort studies to determine whether these approaches provide sus- reflect comparable trends (data not shown). tained clinical improvement in ALS. Of the 15 subjects in the phase 1 trial, 6 subjects Discussion died of their disease and 1 subject died of a congenital In this completed FDA-approved phase 1 trial, 18 intraspi- heart defect unrelated to ALS between 7 and 30 months nal transplantation surgeries in 15 ALS subjects were per- after surgery. Of the 8 subjects who are still alive, 3 of formed following a risk escalation paradigm, progressing them (Subjects 2, 8, and 10) had a long disease course from nonambulatory to ambulatory subjects, lumbar to prior to surgery (5.6, 11.6, and 12.7 years of known dis- cervical spinal cord segments, and unilateral to bilateral ease), likely representing atypical ALS, and have had little injections across 5 cohorts. The encouraging interim change in the trajectory of their disease. Subjects 7, 11, 370 Volume 75, No. 3 Feldman et al.: ALS Stem Cell Transplantation FIGURE 5: Preliminary analysis of potential windows of human spinal stem cell (HSSC) biological activity in Subjects 10 to 12. To identify the most biologically active period of the injected HSSCs, postsurgery data points for Group E subjects were divided into a series of 9-month windows, beginning each month postsurgery, and slopes were calculated across each window. Slopes were also calculated using Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) data points for the presurgical window. (A) The top panel demonstrates ALSFRS-R scores for Group E subjects during the presurgical period (green) and representative ranges associated with the various sliding postsurgical 9-month windows (dark blue). The bottom panel demonstrates the slopes obtained for each sliding window, with the x-axis corresponding to the first month for each 9- month window (ie, window 1 corresponds to months 1–10 postsurgery, window 2 corresponds to months 2–11 postsurgery, window 3 corresponds to months 3–12 postsurgery, etc). The first plotted slope for each subject corresponds to their presurgi- cal progression rate. Slope values higher than the presurgical slope at baseline represent improved or attenuated progression rates during the designated window. Note that the starting month of the final sliding window for each patient coincides with the dates of the second surgery, which occur at 17.5, 19, and 16.6 months after the initial Cohort C surgery (time 0) for Sub- jects 10, 11, and 12, respectively. (B) The presurgical slope and postsurgical slopes associated with the window correlating to the peak benefit windows for both the lumbar and cervical postsurgery time frames are summarized. 12, and 15, who are alive with very slowly progressive or slopes to post-transplant data revealed that >50% of sub- stabilized disease, had 2 clinical characteristics in com- jects demonstrated improvement across multiple clinical mon: these individuals had no bulbar features of ALS, measures at 6, 9, 12, and 15 months postsurgery. Look- and surgical transplantation occurred early within the ing specifically at ALSFRS-R scores at the 9-month time course of their disease (average of 2 years and 1 month point, the subjects who demonstrated improvements were after symptom onset at the time of surgery). These pre- part of Groups B, C, and E and exhibited an average dis- liminary results raise the possibility that intraspinal stem ease duration <2 years prior to surgery, again suggesting cell transplantation of ALS subjects with no bulbar symp- that only subjects early in the disease course may experi- toms early in the course of their disease could slow dis- ence clinical benefit. However, our experience did dem- ease progression or even allow for functional onstrate a wide variation in presurgical progression rates improvement. for those individuals with multiple data points, empha- The majority of ALS trials utilize subject survival sizing the importance of sufficient lead-in data to deter- and ALSFRS-R scores for primary outcome measures. mine efficacy. Average declines of 21.1 ALSFRS-R score Our data demonstrate that GST most closely correlates per month (213.32 per year) have been reported ; how- with the ALSFRS-R scores. Comparisons of presurgical ever, the varied slopes we observed and the heterogeneous March 2014 371 ANNALS of Neurology E.L.F., N.M.B., S.B.R.), the ALS Association (E.L.F.), presentation of ALS emphasize the need for subject- and Neuralstem. Additional support for tissue and data specific baseline data. We acknowledge that this study was not powered analysis was provided by the NIH National Institute on Aging (5P50AG025688; J.D.G.) and the A. Alfred Taub- to determine efficacy and there was no control arm. In man Medical Research Institute (E.L.F., N.M.B.). addition, some subjects exhibited a significant disease burden prior to surgery and were unlikely to show bene- We thank the study participants and their families for their trust and dedication to advancing the field of ALS fit, sufficient preclinical data points were unavailable for therapeutics; the trial Data Safety Monitoring Board, some subjects, and best-fit presurgical slopes were not always significantly powered given the number of avail- chaired by Dr Z. Simmons; Dr T. G. Hazel for provid- able data points. Despite these limitations, we were able ing the CGMP stem cells; L. Shaw for help with subject assessments; Dr M. Gearing for assistance with neuropa- to identify potential possible therapeutic windows in our thology; the staff of the Emory ALS Center; and J. Duell advanced evaluation of Group C=E outcome data. Of note, the 3 subjects in this cohort received the highest for assistance with table preparation. number of injections and demonstrated the largest effects Authorship on progression rates, suggesting that more injections are better, consistent with the neuroprotective mechanism of E.L.F., N.M.B., and J.D.G. designed the study. K.J. pro- 7,9 action hypothesized for this cell therapy. The ability to vided critical study design input. N.M.B., T.F., M.P., successfully administer 1.5 million HSSCs to ALS sub- J.B., and J.D.G. contributed to data acquisition. E.L.F., jects over 15 total injections in Group E subjects into N.M.B., J.H., S.B.R., S.A.S., and J.D.G. were responsi- both lumbar and cervical spinal cord segments over the ble for data analysis and interpretation. E.L.F. and S.A.S. course of 2 surgeries is an important first step in evaluat- drafted the manuscript. All authors critically edited the ing the tolerance of the spinal cord for multiple HSSC content of the article and approved the final version. transplantation procedures. The observed bimodal distri- Potential Conflicts of Interest bution in the 9-month sliding window slope analysis sug- gests there are maximal periods of benefit that correlate N.M.B.: consultancy, Neuralstem; share of sale of Cleve- with the 2 surgical interventions. Furthermore, as the land Clinic Foundation subsidiary IntElect to Boston Sci- bell-shaped benefit curve associated with each interven- entific; patents, licensed to Neuralstem. K.J.: stock, tion is likely due to disease progression, increasing the Neuralstem; patents, assigned to Neuralstem. S.B.R.: total cell dose and applying multiple applications of the equity, consultancy, scientific advisor, Board of Directors, stem cells may increase both the length and magnitude Skulpt; patents, assigned to Skulpt. of potential benefit. These very preliminary observations on only 3 subjects provide the framework for future dis- cussions of trial designs. References In conclusion, as we move forward, the continued assessment of data collected from subjects participating 1. Lunn JS, Sakowski SA, Hur J, Feldman EL. Stem cell technology for neurodegenerative diseases. Ann Neurol 2011;70:353–361. in phase 1 of the trial, evaluation of postsurgical MRI 2. Hefferan MP, Galik J, Kakinohana O, et al. Human neural stem data, and characterization of the cellular grafts in cell replacement therapy for amyotrophic lateral sclerosis by spinal deceased subjects will provide further insight into the transplantation. PLoS One 2012;7:e42614. therapeutic mechanisms and potential efficacy of intraspi- 3. Xu L, Ryugo DK, Pongstaporn T, et al. Human neural stem cell nal stem cell transplantation in ALS. 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March 2014 373

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Annals of NeurologyPubmed Central

Published: Mar 7, 2014

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