Methylation-Sensitive Restriction Enzyme Quantitative Polymerase Chain Reaction Enables Rapid, Accurate, and Precise Detection of Methylation Status of the Regulatory T Cell (Treg)-Specific Demethylation Region in Primary Human Tregs.

Human regulatory T cells (Tregs) have been implicated in cancer immunotherapy and are also an emerging cellular therapeutic for the treatment of multiple indications. Although Treg stability during ex vivo culture has improved, methods to assess Treg stability such as bisulfite Sanger sequencing to determine the methylation status of the Treg-specific demethylated region (TSDR) have remained unchanged. Bisulfite Sanger sequencing is not only costly and cumbersome to perform, it is inaccurate because of relatively low read counts. Bisulfite next-generation sequencing, although more accurate, is a less accessible method. In this study, we describe the application of methylation-sensitive restriction enzymes (MSRE) and quantitative PCR (qPCR) to determine the methylation status of the TSDR. Using known ratios of Tregs and non-Tregs, we show that MSRE-qPCR can distinguish the methylation status of the TSDR in populations of cells containing increasing proportions of Tregs from 0 to 100%. In a comparison with values obtained from an established bisulfite next-generation sequencing approach for determining the methylation status of the TSDR, our MSRE-qPCR results were within 5% on average for all samples with a high percentage (>70%) of Tregs, reinforcing that MSRE-qPCR can be completed in less time than other methods with the same level of accuracy. The value of this assay was further demonstrated by quantifying differences in TSDR methylation status of Tregs treated with and without rapamycin during an ex vivo expansion culture. Together, we show that our novel application of the MSRE-qPCR to the TSDR is an optimal assay for accurate assessment of Treg purity.

In Situ Modification of Tissue Stem and Progenitor Cell Genomes.

In vivo delivery of genome-modifying enzymes holds significant promise for therapeutic applications and functional genetic screening. Delivery to endogenous tissue stem cells, which provide an enduring source of cell replacement during homeostasis and regeneration, is of particular interest. Here, we use a sensitive Cre/lox fluorescent reporter system to test the efficiency of genome modification following in vivo transduction by adeno-associated viruses (AAVs) in tissue stem and progenitor cells. We combine immunophenotypic analyses with in vitro and in vivo assays of stem cell function to reveal effective targeting of skeletal muscle satellite cells, mesenchymal progenitors, hematopoietic stem cells, and dermal cell subsets using multiple AAV serotypes. Genome modification rates achieved through this system reached >60%, and modified cells retained key functional properties. This study establishes a powerful platform to genetically alter tissue progenitors within their physiological niche while preserving their native stem cell properties and regulatory interactions.

Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

Amelioration of Alpha-1 Antitrypsin Deficiency Diseases with Genome Editing in Transgenic Mice.

Alpha-1 antitrypsin deficiency (AATD) is a hereditary liver disease caused by mutations in the SERPINA1 serine protease inhibitor gene. Most severe patients are homozygous for PiZ alleles (PiZZ; amino acid E324K), which lead to protein aggregates in hepatocytes and reduced circulating levels of AAT. The liver aggregates typically lead to fibrosis, cirrhosis, and hepatocellular carcinoma, and the reduced circulating AAT levels can lead to emphysema and chronic obstructive pulmonary diseases. In this study, two CRISPR/Cas9 gene editing approaches were used to decrease liver aggregates and increase systemic AAT-M levels in the PiZ transgenic mouse. In the first approach, AAT expression in hepatocytes was reduced more than 98% following the systemic delivery of AAV8-CRISPR targeting exon 2 of hSERPINA1, leading to reduced aggregates in hepatocytes. In the second approach, a second adeno-associated virus, which provided the donor template to correct the Z mutation, was also administered. These treated mice had reduced AAT expression (> 98%) and a low level (5%) of wildtype AAT-M mRNA. Taken together, this study shows that CRISPR gene editing can efficiently reduce liver expression of AAT-Z and restore modest levels of wildtype AAT-M in a mouse model of AATD, raising the possibility of CRISPR gene editing therapeutic for AATD.

Synergistic inhibition of Aß production by combinations of γ-secretase modulators.

Alzheimer's disease is associated with the accumulation of amyloid-ß (Aß) in the brain. In particular, the 42-amino acid form, Aß1-42, is thought to play a key role in the disease. It is therefore of interest that diverse compounds, known as γ-secretase modulators (GSM), can selectively decrease Aß1-42 production without inhibiting the production of other forms of Aß. Here we describe the novel discovery of synergistic inhibition of Aß by certain combinations of GSMs. Cell cultures were treated with pairwise combinations of GSMs to determine how Aß peptide production was affected. Analysis of isobolograms and calculation of the combination index showed that BMS-869780 and GSM-2 were highly synergistic. Additional combinations of GSMs revealed that inhibition of Aß occurred only when one GSM was of the "acid GSM" structural class and the other was of the "non-acid GSM" class. A total of 15 representative acid/non-acid GSM combinations were shown to inhibit Aß production, whereas 10 pairwise combinations containing two acid GSMs or containing two non-acid GSMs did not inhibit Aß. We also discovered that lasalocid, a natural product, is a potent GSM. Lasalocid is unique in that it did not synergize with other GSMs. Synergism did not translate in vivo perhaps because of biochemical differences between the cell culture model and brain. These findings reinforce the pharmacological differences between different structural classes of GSMs, and may help to exploit the potential of γ-secretase as a drug target.

Targeting Prodromal Alzheimer Disease With Avagacestat: A Randomized Clinical Trial.

IMPORTANCE: Early identification of Alzheimer disease (AD) is important for clinical management and affords the opportunity to assess potential disease-modifying agents in clinical trials. To our knowledge, this is the first report of a randomized trial to prospectively enrich a study population with prodromal AD (PDAD) defined by cerebrospinal fluid (CSF) biomarker criteria and mild cognitive impairment (MCI) symptoms. OBJECTIVES: To assess the safety of the γ-secretase inhibitor avagacestat in PDAD and to determine whether CSF biomarkers can identify this patient population prior to clinical diagnosis of dementia. DESIGN, SETTING, AND PARTICIPANTS: A randomized, placebo-controlled phase 2 clinical trial with a parallel, untreated, nonrandomized observational cohort of CSF biomarker-negative participants was conducted May 26, 2009, to July 9, 2013, in a multicenter global population. Of 1358 outpatients screened, 263 met MCI and CSF biomarker criteria for randomization into the treatment phase. One hundred two observational cohort participants who met MCI criteria but were CSF biomarker-negative were observed during the same study period to evaluate biomarker assay sensitivity. INTERVENTIONS: Oral avagacestat or placebo daily. MAIN OUTCOMES AND MEASURE: Safety and tolerability of avagacestat. RESULTS: Of the 263 participants in the treatment phase, 132 were randomized to avagacestat and 131 to placebo; an additional 102 participants were observed in an untreated observational cohort. Avagacestat was relatively well tolerated with low discontinuation rates (19.6%) at a dose of 50 mg/d, whereas the dose of 125 mg/d had higher discontinuation rates (43%), primarily attributable to gastrointestinal tract adverse events. Increases in nonmelanoma skin cancer and nonprogressive, reversible renal tubule effects were observed with avagacestat. Serious adverse event rates were higher with avagacestat (49 participants [37.1%]) vs placebo (31 [23.7%]), attributable to the higher incidence of nonmelanoma skin cancer. At 2 years, progression to dementia was more frequent in the PDAD cohort (30.7%) vs the observational cohort (6.5%). Brain atrophy rate in PDAD participants was approximately double that of the observational cohort. Concordance between abnormal amyloid burden on positron emission tomography and pathologic CSF was approximately 87% (κ = 0.68; 95% CI, 0.48-0.87). No significant treatment differences were observed in the avagacestat vs placebo arm in key clinical outcome measures. CONCLUSIONS AND RELEVANCE: Avagacestat did not demonstrate efficacy and was associated with adverse dose-limiting effects. This PDAD population receiving avagacestat or placebo had higher rates of clinical progression to dementia and greater brain atrophy compared with CSF biomarker-negative participants. The CSF biomarkers and amyloid positron emission tomography imaging were correlated, suggesting that either modality could be used to confirm the presence of cerebral amyloidopathy and identify PDAD. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00890890.

Pharmacodynamics of selective inhibition of γ-secretase by avagacestat.

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid ß-peptide (Aß), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aß and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aß and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aß levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aß40 and Aß42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending-dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aß40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aß levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.

Safety and tolerability of the γ-secretase inhibitor avagacestat in a phase 2 study of mild to moderate Alzheimer disease.

OBJECTIVE: To assess the safety, tolerability, and pharmacokinetic and pharmacodynamic effects of the -secretase inhibitor avagacestat in patients with mild to moderate Alzheimer disease (AD). DESIGN: Randomized, double-blind, placebo-controlled,24-week phase 2 study. SETTING: Global, multicenter trial. PATIENTS: A total of 209 outpatients with mild to moderate AD were randomized into the double-blind treatment phase. The median age of the patients was 75 years,58.9% were APOE ε4 carriers, and baseline measures of disease severity were similar among groups. INTERVENTION: Avagacestat, 25, 50, 100, or 125 mg daily,or placebo administered orally daily. MAIN OUTCOME MEASURES: Safety and tolerability of avagacestat. RESULTS: Discontinuation rates for the 25-mg and 50-mg doses of avagacestat were comparable with placebo but were higher in the 100-mg and 125-mg dose groups.Trends for worsening cognition, as measured by change from baseline Alzheimer Disease Assessment Scale cognitive subscale score, were observed in the 100-mg and125-mg dose groups. Treatment-emergent serious adverse events were similar across placebo and treatment groups. The most common reason for discontinuation was adverse events, predominantly gastrointestinal anddermatologic. Other adverse events occurring more frequentlyin patients undergoing treatment included reversibleglycosuria (without associated serum glucose changes), nonmelanoma skin cancer, and asymptomaticmagnetic resonance imaging findings. Exploratory cerebrospinal fluid amyloid isoforms and tau biomarker analysis demonstrated dose-dependent but not statistically significant reductions in a small subset of patients. CONCLUSIONS: Avagacestat dosed at 25 and 50 mg daily was relatively well tolerated and had low discontinuation rates. The 100-mg and 125-mg dose arms were poorly tolerated with trends for cognitive worsening. Exploratory cerebrospinal fluid biomarker substudies provide preliminary support for -secretase target engagement,but additional studies are warranted to better characterize pharmacodynamic effects at the 25- and 50-mg doses.This study establishes an acceptable safety and tolerability dose range for future avagacestat studies in AD. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00810147

Multicenter, randomized, double-blind, placebo-controlled, single-ascending dose study of the oral γ-secretase inhibitor BMS-708163 (Avagacestat): tolerability profile, pharmacokinetic parameters, and pharmacodynamic markers.

BACKGROUND: γ-Secretase inhibitors (GSIs) are being investigated for their potential to modify the progression of Alzheimer disease based on their ability to regulate amyloid-ß (Aß) accumulation. BMS-708163 (avagacestat) is an oral GSI designed for selective inhibition of Aß synthesis currently in development for the treatment of mild to moderate and predementia AD. In addition to the desired effect on Aß synthesis, GSIs affect Notch processing, which is thought to mediate some toxic adverse effects reported with this drug class. Avagacestat produced up to 190-fold greater selectivity for Aß synthesis than Notch processing in preclinical studies and may therefore produce less toxic adverse events than other less selective compounds. Presented here are the results of the first in-human study for this new GSI compound. OBJECTIVE: The goal of this study was to assess the tolerability profile, pharmacokinetic properties, and effects on pharmacodynamic markers (Aß, trefoil factor family 3 protein, dual specificity phosphatase 6, and hairy and enhancer of split-1) of single, oral doses of avagacestat in healthy, young, male volunteers. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled, single-ascending dose study in 8 healthy young men (age, 18-45 years) per dosing panel. Each study participant was randomized to receive a single dose of placebo (n = 2) or avagacestat (n = 6 for each dose) as an oral solution in 1 of 9 sequential dose panels (0.3, 1.5, 5, 15, 50, 100, 200, 400, and 800 mg). For determination of avagacestat, blood samples were obtained before dosing and for up to 144 hours after dosing. For participants in the 800-mg avagacestat dose panel, additional samples were obtained at 216, 312, and 648 hours. For 40-amino acid isoform of Aß (Aß(1-40)) assessment, plasma samples were collected before avagacestat administration and up to 72 hours after dosing. RESULTS: Avagacestat concentrations peaked quickly after oral administration and then had a biphasic decrease in concentrations with a prolonged terminal phase. Exposures were proportional with doses up to 200 mg. Avagacestat was well tolerated at single oral doses up to 800 mg, with a biphasic effect on plasma Aß(1-40). Adverse events were predominately mild to moderate in severity with no evidence of dose dependence up to 200 mg. CONCLUSIONS: Results from this single-ascending dose study suggest that avagacestat was tolerated at a single-dose range of 0.3 to 800 mg and suitable for further clinical development.

Discovery of matrix metalloproteases selective and activated peptide-doxorubicin prodrugs as anti-tumor agents.

To selectively target doxorubicin (Dox) to tumor tissue and thereby improve the therapeutic index and/or efficacy of Dox, matrix metalloproteinases (MMP) activated peptide-Dox prodrugs were designed and synthesized by coupling MMP-cleavable peptides to Dox. Preferred conjugates were good substrates for MMPs, poor substrates for neprilysin, an off-target proteinase, and stable in blood ex vivo. When administered to mice with HT1080 xenografts, conjugates, such as 19, preferentially released Dox in tumor relative to heart tissue and prevented tumor growth with less marrow toxicity than Dox.

[3H]BMS-599240--a novel tritiated ligand for the characterization of BACE1 inhibitors.

In this report we describe a novel radioligand, [(3)H](S)-2-((S)-3-Acetylamino-3-sec-butyl-2-oxo-pyrrolidin-1-yl)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-methoxy-benzylamino)-propyl]-4-phenyl-butyramide ([(3)H]BMS-599240), that exhibits robust specific binding in homogenates from cell cultures overexpressing beta-site amyloid precursor protein cleaving enzyme-1 (BACE1). Radioligand binding exhibited high affinity, K(d)=2 nM, commensurate with its inhibitory potency against BACE1. Inhibition of radioligand binding in the presence of a range of different BACE1 inhibitors exhibited the same rank order of potency as for inhibition of BACE1 enzymatic activity. BACE1-dependent binding of the radioligand was also demonstrated in mouse brain homogenates, where genetic ablation of BACE1 eliminated high affinity binding. Thus, the radioligand [(3)H]BMS-599240 is a novel tool potentially useful for evaluation of BACE1 enzyme in biological samples, and for evaluation of inhibitor binding to BACE1.

Recent progress in the medicinal chemistry of gamma-secretase inhibitors.

Abeta is implicated in the initiation and progression of Alzheimer's disease (AD) by the phenotypic analysis of mutations in three human genes that lead to inherited, early forms of AD and data from preclinical studies. Based on this evidence, gamma-secretase inhibitors are being actively pursued as potential AD therapeutics to reduce Abeta formation. This manuscript reviews recent progress in the medicinal chemistry of three major classes of gamma-secretase inhibitors: peptide isosteres, azepines, and sulfonamides. Peptide isosteres have been useful for demonstrating that presenilin is the catalytic subunit of gamma-secretase and probing the active site. The peptidic nature of these inhibitors has, however, interfered with their utility for in vivo studies. Instead, the pharmaceutical industry has focused on optimizing azepines and sulfonamides. Both azepines and sulfonamides bind to a common, allosteric site on presenilin that differs from the active site identified by the peptide isosteres. Significant progress in the optimization of both azepines and sulfonamides has led to compounds that inhibit brain Abeta synthesis in preclinical models and has culminated in the identification of gamma-secretase inhibitors, including LY-450139 and MK-0752, for human trials.

Matrix metalloproteinase-activated doxorubicin prodrugs inhibit HT1080 xenograft growth better than doxorubicin with less toxicity.

Matrix metalloproteinase (MMP)-activated prodrugs were formed by coupling MMP-cleavable peptides to doxorubicin. The resulting conjugates were excellent in vitro substrates for MMP-2, -9, and -14. HT1080, a fibrosarcoma cell line, was used as a model system to test these prodrugs because these cells, like tumor stromal fibroblasts, expressed several MMPs. In cultured HT1080 cells, simple MMP-cleavable peptides were primarily metabolized by neprilysin, a membrane-bound metalloproteinase. MMP-selective metabolism in cultured HT1080 cells was obtained by designing conjugates that were good MMP substrates but poor neprilysin substrates. To determine how conjugates were metabolized in animals, MMP-selective conjugates were given to mice with HT1080 xenografts and the distribution of doxorubicin was determined. These studies showed that MMP-selective conjugates were preferentially metabolized in HT1080 xenografts, relative to heart and plasma, leading to 10-fold increases in the tumor/heart ratio of doxorubicin. The doxorubicin deposited by a MMP-selective prodrug, compound 6, was more effective than doxorubicin at reducing HT1080 xenograft growth. In particular, compound 6 cured 8 of 10 mice with HT1080 xenografts at doses below the maximum tolerated dose, whereas doxorubicin cured 2 of 20 mice at its maximum tolerated dose. Compound 6 was less toxic than doxorubicin at this efficacious dose because mice treated with compound 6 had no detectable changes in body weight or reticulocytes, a marker for marrow toxicity. Hence, MMP-activated doxorubicin prodrugs have a much higher therapeutic index than doxorubicin using HT1080 xenografts as a preclinical model.

Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display.

The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) downward arrow (L/Y)(T/V/M/R)(R/K), where downward arrow denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E downward arrow LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest kcat/Km values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower kcat/Km values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer.

Expression, purification, and kinetic characterization of full-length human fibroblast activation protein.

Human fibroblast activation protein (FAP), an integral membrane serine protease, was produced in insect cells as a hexa-His-tagged protein using a recombinant baculovirus expression system. Two isoforms of FAP, glycosylated and nonglycosylated, were identified by Western blotting using an anti-His-tag antibody and separated by lectin chromatography. The glycosylated FAP was purified to near homogeneity using immobilized metal affinity chromatography and was shown to have both postprolyl dipeptidyl peptidase and postgelatinase activities. In contrast, the nonglycosylated isoform demonstrated no detectable gelatinase activity by either zymography or a fluorescence-based gelatinase activity assay. The kinetic parameters of the dipeptidyl peptidase activity for glycosylated FAP were determined using dipeptide Ala-Pro-7-amino-trifluoromethyl-coumarin as the substrate. The k(cat) is 2.0 s(-1) and k(cat)/K(m) is 1.0 x 10(4) M(-1) s(-1) at pH 8.5. The pH dependence of k(cat) reveals two ionization groups with pK(a1) of 7.0 and pK(a2) of 11.0. The pH profile of k(cat)/K(m) yields similar results with pK(a1) 6.2 and pK(a2) 11.0. The neutral pK(a1) is associated with His at the active site. The basic pK(a2) might be contributed from an ionization group that is not involved directly in catalysis, instead associated with the stability of the active site structure.