Safety and efficacy of first-in-man intrathecal injection of human astrocytes (AstroRx®) in ALS patients: phase I/IIa clinical trial results.

BACKGROUND: Malfunction of astrocytes is implicated as one of the pathological factors of ALS. Thus, intrathecal injection of healthy astrocytes in ALS can potentially compensate for the diseased astrocytes. AstroRx® is an allogeneic cell-based product, composed of healthy and functional human astrocytes derived from embryonic stem cells. AstroRx® was shown to clear excessive glutamate, reduce oxidative stress, secrete various neuroprotective factors, and act as an immunomodulator. Intrathecal injection of AstroRx® to animal models of ALS slowed disease progression and extended survival. Here we report the result of a first-in-human clinical study evaluating intrathecal injection of AstroRx® in ALS patients. METHODS: We conducted a phase I/IIa, open-label, dose-escalating clinical trial to evaluate the safety, tolerability, and therapeutic effects of intrathecal injection of AstroRx® in patients with ALS. Five patients were injected intrathecally with a single dose of 100 × 106 AstroRx® cells and 5 patients with 250 × 106 cells (low and high dose, respectively). Safety and efficacy assessments were recorded for 3 months pre-treatment (run-in period) and 12 months post-treatment (follow-up period). RESULTS: A single administration of AstroRx® at either low or high doses was safe and well tolerated. No adverse events (AEs) related to AstroRx® itself were reported. Transient AEs related to the Intrathecal (IT) procedure were all mild to moderate. The study demonstrated a clinically meaningful effect that was maintained over the first 3 months after treatment, as measured by the pre-post slope change in ALSFRS-R. In the 100 × 106 AstroRx® arm, the ALSFRS-R rate of deterioration was attenuated from - 0.88/month pre-treatment to - 0.30/month in the first 3 months post-treatment (p = 0.039). In the 250 × 106 AstroRx® arm, the ALSFRS-R slope decreased from - 1.43/month to - 0.78/month (p = 0.0023). The effect was even more profound in a rapid progressor subgroup of 5 patients. No statistically significant change was measured in muscle strength using hand-held dynamometry and slow vital capacity continued to deteriorate during the study. CONCLUSIONS: Overall, these findings suggest that a single IT administration of AstroRx® to ALS patients at a dose of 100 × 106 or 250 × 106 cells is safe. A signal of beneficial clinical effect was observed for the first 3 months following cell injection. These results support further investigation of repeated intrathecal administrations of AstroRx®, e.g., every 3 months. TRIAL REGISTRATION: NCT03482050.

Safety and efficacy of human embryonic stem cell-derived astrocytes following intrathecal transplantation in SOD1G93A and NSG animal models.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease characterized by the loss of MNs in the central nervous system. As MNs die, patients progressively lose their ability to control voluntary movements, become paralyzed and eventually die from respiratory/deglutition failure. Despite the selective MN death in ALS, there is growing evidence that malfunctional astrocytes play a crucial role in disease progression. Thus, transplantation of healthy astrocytes may compensate for the diseased astrocytes. METHODS: We developed a good manufacturing practice-grade protocol for generation of astrocytes from human embryonic stem cells (hESCs). The first stage of our protocol is derivation of astrocyte progenitor cells (APCs) from hESCs. These APCs can be expanded in large quantities and stored frozen as cell banks. Further differentiation of the APCs yields an enriched population of astrocytes with more than 90% GFAP expression (hES-AS). hES-AS were injected intrathecally into hSOD1G93A transgenic mice and rats to evaluate their therapeutic potential. The safety and biodistribution of hES-AS were evaluated in a 9-month study conducted in immunodeficient NSG mice under good laboratory practice conditions. RESULTS: In vitro, hES-AS possess the activities of functional healthy astrocytes, including glutamate uptake, promotion of axon outgrowth and protection of MNs from oxidative stress. A secretome analysis shows that these hES-AS also secrete several inhibitors of metalloproteases as well as a variety of neuroprotective factors (e.g. TIMP-1, TIMP-2, OPN, MIF and Midkine). Intrathecal injections of the hES-AS into transgenic hSOD1G93A mice and rats significantly delayed disease onset and improved motor performance compared to sham-injected animals. A safety study in immunodeficient mice showed that intrathecal transplantation of hES-AS is safe. Transplanted hES-AS attached to the meninges along the neuroaxis and survived for the entire duration of the study without formation of tumors or teratomas. Cell-injected mice gained similar body weight to the sham-injected group and did not exhibit clinical signs that could be related to the treatment. No differences from the vehicle control were observed in hematological parameters or blood chemistry. CONCLUSION: Our findings demonstrate the safety and potential therapeutic benefits of intrathecal injection of hES-AS for the treatment of ALS.

A fluorescent multiplex-DGGE screening test for mutations in the BRCA1 gene.

Screening for mutations in the BRCA1 gene is challenging because of the wide spectrum of mutations found in this large gene. As the extensive exon 11 is commonly screened by the protein truncation test (PTT), here a fluorescent multiplex denaturing gradient gel electrophoresis (FMD) mutation screening technique was developed to test the remaining numerous small exons and splice sites of the gene. The method is based upon the use of an efficient multiplex polymerase chain reaction (PCR) amplification of the target regions, followed by denaturing gradient gel electrophoresis (DGGE) separation of the amplicon mixture, and the immediate achievement of results by wet gel scanning. The technique was applied to screen 16 samples with different BRCA1 sequence variants distributed over 12 exons. All variants were detected. In addition, 188 DNA samples from ovarian cancer patients were screened, identifying 22 new sequence variants (11.7% of the samples) and 243 common polymorphisms in the BRCA1 locus. Variants included 16 single nucleotide substitutions, 3 deletions of 2 nucleotides, 1 deletion of 4 nucleotides, and 2 insertions of 1 nucleotide. The FMD test provides an accurate, fast, nonradioactive and cost-efficient way to scan the BRCA1 gene with high sensitivity and an ease of result interpretation. This technique may prove to be a useful research tool for the detection of mutations and polymorphisms in the BRCA1 gene and for large-scale epidemiologic studies.

Mutation screening using fluorescence multiplex denaturing gradient gel electrophoresis (FMD): detecting mutations in the BRCA1 gene.

Fluorescent multiplex denaturing gradient gel electrophoresis (FMD) is a mutation screening technique designed to detect unknown as well as previously identified mutations. FMD constitutes a recent modification of the standard denaturing gradient gel electrophoresis (DGGE) technique, which combines multiplex PCR amplification of target DNA using fluorescently labeled primers with DGGE separation of the amplicon mixture, allowing immediate identification of sequence variants by wet gel scanning. FMD permits the simultaneous detection of small insertions, deletions and single nucleotide substitutions among multiple DNA fragments (up to 480 fragments) from 96 samples in parallel for each run. It increases output and reduces cost dramatically compared with classical DGGE, without sacrificing sensitivity and accuracy in detecting mutations. This protocol details an accurate, fast, nonradioactive and cost-effective way to screen the BRCA1 gene for mutations with high sensitivity, providing easily interpreted results. It may also be adapted to screen other target genes and/or used in large-scale epidemiological studies.

Cancer incidence in a population of Jewish women at risk of ovarian cancer.

PURPOSE: To evaluate the incidence and clinical characteristics of ovarian and other cancers in a cohort of women at risk of developing ovarian cancer. PATIENTS AND METHODS: The Gilda Radner Ovarian Cancer Detection Program in Los Angeles, CA, was established in 1991 to study the efficacy of screening in the early detection of ovarian cancer. We present findings from a historical cohort of 290 Jewish women who were offered BRCA testing for three common founder mutations (BRCA1 185delAG and 5382insC and BRCA2 6174delT). RESULTS: In 10 years, 17 cancers were observed (1,111 per 100,000 per year), including six breast and eight ovarian or related cancers. A high proportion of cancers of peritoneal origin was observed. The majority (86%) of women with incident breast or ovarian/peritoneal cancer carried a mutation in the BRCA1 gene. The overall cancer incidence among carriers of mutations in the BRCA1 gene was estimated to be 5,450 per 100,000 per year, corresponding to a cumulative incidence of 47.5% at 10 years. In contrast, the cumulative incidence of cancer among noncarriers was 2.5% (P < 10(-8)). After adjustment for sampling, the risks to BRCA1 mutation carriers at 10 years were estimated to be 21% for ovarian/peritoneal/tubal cancer, 16% for breast cancer, and 36% for all cancers. CONCLUSION: The excess risk of breast and ovarian cancer in Jewish women with a family history of ovarian cancer is largely attributable to mutations in BRCA1. Intensive surveillance by use of CA-125 and ultrasound does not seem to be an effective means of diagnosing early-stage ovarian cancer in this high-risk cohort.