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Gene-editing technologies, which include the CRISPR–Cas nucleases1–3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a ‘once-and-done’ approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs. In a cynomolgus macaque model, CRISPR base editors delivered in lipid nanoparticles are shown to efficiently and stably knock down PCSK9 in the liver to reduce levels of PCSK9 and low-density lipoprotein cholesterol in the blood.

Novel nanoparticle-drug conjugates (NDCs) containing diverse, clinically relevant anticancer drug payloads (docetaxel, cabazitaxel, and gemcitabine) were successfully generated and tested in drug discovery studies. The NDCs utilized structurally varied linkers that attached the drug payloads to a β-cyclodextrin-PEG copolymer to form self-assembled nanoparticles. In vitro release studies revealed a diversity of release rates driven by linker structure-activity relationships (SARs). Improved in vivo pharmacokinetics (PK) for the cabazitaxel (CBTX) NDCs with glycinate-containing (1c) and hexanoate-containing linkers (2c) were demonstrated, along with high and sustained tumor levels (>168 h of released drug in tumor tissues). This led to potent efficacy and survival in both taxane- and docetaxel-resistant in vivo anticancer mouse efficacy models. Overall, the CBTX-hexanoate NDC 2c (CRLX522), demonstrated optimal and improved in vivo PK (plasma and tumor) and efficacy profile versus those of the parent drug, and the results support the potential therapeutic use of CRLX522 as a new anticancer agent.

Summary: The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform. : Finn et al. describe the development of a transient, biodegradable LNP-based CRISPR/Cas9 delivery system that achieves >97% knockdown of serum TTR levels following a single administration. Editing levels were stable for 12 months, despite the transient nature of the delivery system and the editing components. Keywords: CRISPR, Cas9, genome editing, LNP, lipid nanoparticle, TTR, CRISPR/Cas9, liver delivery, gene therapy, sgRNA

VEGF pathway–targeting antiangiogenic drugs, such as bevacizumab, when combined with chemotherapy have changed clinical practice for the treatment of a broad spectrum of human cancers. However, adaptive resistance often develops, and one major mechanism is elevated tumor hypoxia and upregulated hypoxia-inducible factor-1α (HIF1α) caused by antiangiogenic treatment. Reduced tumor vessel numbers and function following antiangiogenic therapy may also affect intratumoral delivery of concurrently administered chemotherapy. Nonetheless, combining chemotherapy and bevacizumab can lead to improved response rates, progression-free survival, and sometimes, overall survival, the extent of which can partly depend on the chemotherapy backbone. A rational, complementing chemotherapy partner for combination with bevacizumab would not only reduce HIF1α to overcome hypoxia-induced resistance, but also improve tumor perfusion to maintain intratumoral drug delivery. Here, we evaluated bevacizumab and CRLX101, an investigational nanoparticle–drug conjugate containing camptothecin, in preclinical mouse models of orthotopic primary triple-negative breast tumor xenografts, including a patient-derived xenograft. We also evaluated long-term efficacy of CRLX101 and bevacizumab to treat postsurgical, advanced metastatic breast cancer in mice. CRLX101 alone and combined with bevacizumab was highly efficacious, leading to complete tumor regressions, reduced metastasis, and greatly extended survival of mice with metastatic disease. Moreover, CRLX101 led to improved tumor perfusion and reduced hypoxia, as measured by contrast-enhanced ultrasound and photoacoustic imaging. CRLX101 durably suppressed HIF1α, thus potentially counteracting undesirable effects of elevated tumor hypoxia caused by bevacizumab. Our preclinical results show pairing a potent cytotoxic nanoparticle chemotherapeutic that complements and improves concurrent antiangiogenic therapy may be a promising treatment strategy for metastatic breast cancer. Cancer Res; 76(15); 4493–503. ©2016 AACR.

Reducing the production of larger aggregation-prone amyloid β-peptides (Aβ) remains an untested therapeutic approach for reducing the appearance and growth of Aβ plaques in the brain, which are a hallmark pathological feature of Alzheimer's disease. γ-Secretase modulators (GSMs) are therapeutics that impact γ-secretase-dependent cleavage of amyloid precursor protein to promote the production of shorter Aβ peptides that are less prone to aggregation and plaque deposition. This is accomplished without inhibiting overall γ-secretase function and cleavage of other substrates, which is believed to be a source of deleterious side effects. Here, we report the pharmacokinetic and pharmacodynamic properties of BIIB042, a novel bioavailable and brain-penetrant GSM. In cell-based assays, BIIB042 reduced the levels of Aβ42, increased the levels of Aβ38 and had little effect on the levels of Aβ40, the most abundant Aβ species. Similar pharmacodynamic properties were confirmed in the central nervous system and in plasma of mice and rats, and also in plasma of cynomolgus monkeys after a single oral dose of BIIB042. BIIB042 reduced Aβ42 levels and Aβ plaque burden in Tg2576 mice, which overexpress human amyloid precursor protein and serve as a model system for Alzheimer's disease. BIIB042 did not inhibit cleavage of other γ-secretase substrates in cell-based and in vivo signaling and cleavage assays. The pharmacodynamic effects of lowering Aβ42 in the central nervous system coupled with demonstrated efficacy in reducing plaque pathology suggests modulation of γ-secretase, with molecules like BIIB042, is a compelling therapeutic approach for the treatment of Alzheimer's disease.

Protein interacting with C kinase (PICK1) is a scaffolding protein that is present in dendritic spines and interacts with a wide array of proteins through its PDZ domain. The best understood function of PICK1 is regulation of trafficking of AMPA receptors at neuronal synapses via its specific interaction with the AMPA GluA2 subunit. Disrupting the PICK1-GluA2 interaction has been shown to alter synaptic plasticity, a molecular mechanism of learning and memory. Lack of potent, selective inhibitors of the PICK1 PDZ domain has hindered efforts at exploring the PICK1-GluA2 interaction as a therapeutic target for neurological diseases. Here, we report the discovery of PICK1 small molecule inhibitors using a structure-based drug design strategy. The inhibitors stabilized surface GluA2, reduced Aβ-induced rise in intracellular calcium concentrations in cultured neurons, and blocked long term depression in brain slices. These findings demonstrate that it is possible to identify potent, selective PICK1-GluA2 inhibitors which may prove useful for treatment of neurodegenerative disorders.

Many studies have focused on the challenges of small molecule uptake across the blood–brain barrier, whereas few in-depth studies have assessed the challenges with the uptake of antibodies into the central nervous system (CNS). In drug development, cerebrospinal fluid (CSF) sampling is routinely used as a surrogate for assessing CNS drug exposure and biomarker levels. In this report, we have studied the kinetic correlation between CSF and serum drug concentration–time profiles for five humanized monoclonal antibodies in rats and cynomolgus monkeys and analyzed factors that affect their CSF exposure. Upon intravenous (IV) bolus injection, antibodies entered the CNS slowly and reached maximum CSF concentration ( CSF T max ) in one to several days in both rats and monkeys. Antibody serum and CSF concentration–time curves converged until they became parallel after CSF T max was reached. Antibody half-lives in CSF ( CSF t ½ ) approximated their serum half-lives ( serum t ½ ). Although the intended targets of these antibodies were different, the steady-state CSF to serum concentration ratios were similar at 0.1–0.2% in both species. Independent of antibody target and serum concentration, CSF-to-serum concentration ratios for individual monkeys ranged by up to tenfold from 0.03 to 0.3%. Upon systemic administration, average antibodies CSF-to-serum concentration ratios in rats and monkeys were 0.1–0.2%. The CSF t ½ of the antibodies was largely determined by their long systemic t ½ ( systemic t ½ ).

The objectives of the study were to characterize the selectivity of dantrolene to breast cancer resistance protein (Bcrp) and to evaluate whether cerebrospinal fluid (CSF) can be used as a surrogate to assess brain exposures of BCRP and P-glycoprotein (Pgp) substrates. The impact of Bcrp and Pgp on dantrolene exposures in brain and CSF was examined in Bcrp and Mdr1a/1b knockout mice and was further investigated in wild-type mice in the presence of the Bcrp inhibitor (3 S ,6 S ,12a S )-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1′,2′:1,6]pyrido[3,4- b ]indole-3-propanoic acid 1,1-dimethylethyl ester (Ko143), the Pgp inhibitor 6-[(2 S ,4 R ,6 E )-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporine A (PSC833), and the dual inhibitor N -(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). The effect of Bcrp and Pgp on digoxin exposures in brain and CSF was investigated in wild-type mice in the presence of the inhibitors. In vivo studies showed dantrolene exposures in brain and CSF, but not the blood, increased in Bcrp(−/−) and Mdr1a/1b(−/−)/Bcrp(−/−) mice, or in the presence of the Bcrp inhibitors Ko143 or GF120918. Inhibition of Pgp by GF120918 and PSC833 significantly increased digoxin exposures in brain, CSF, and blood to a lesser extent. Results from the present study demonstrated that inhibition of Bcrp and Pgp increased not only the exposures of dantrolene and digoxin in brain, but also the exposures in CSF. In addition, the change of exposures in CSF reflected the changes in brain. The present study strongly suggests that the dantrolene and digoxin exposures in CSF are primarily determined by the rapid transport from brain to CSF, and inhibition of Bcrp and Pgp exhibits little impact on using CSF as surrogates to assess brain exposures of Bcrp and Pgp substrates.

Starting from literature examples of nonsteroidal anti-inflammatory drugs (NSAIDs)-type carboxylic acid γ-secretase modulators (GSMs) and using a scaffold design approach, we identified 4-aminomethylphenylacetic acid 4 with a desirable γ-secretase modulation profile. Scaffold optimization led to the discovery of a novel chemical series, represented by 6b, having improved brain penetration. Further SAR studies provided analog 6q that exhibited a good pharmacological profile. Oral administration of 6q significantly reduced brain Aβ42 levels in mice and rats.

Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (−)-(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydronaphthalen-2-yl) propan-1-ol was identified as a good SphK2 substrate and potent S1P1 agonist with good oral bioavailability.

The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors.

To support in vivo screening efforts for estrogen receptor (ER) subtype selective therapeutic agents, we initiated work to discover surrogate markers (biomarkers) in blood plasma that would change in response to ER subtype-specific action. We used a proteomic approach employing strong anion exchange chromatography (SAX), PAGE, and MS to identify potential plasma markers for selective ER-alpha action. The methodology was used to compare blood from vehicle-treated rats to blood from rats treated with either 17beta-estradiol (an ER-alpha/ER-beta agonist) or compound 1 (17alpha-ethynyl-[3,2-c]pyrazolo-19-nor-4-androstene-17beta-ol, an ER-alpha-selective agonist). Blood samples were first fractionated by SAX to separate fractions containing dominant common plasma proteins from fractions enriched for less-abundant plasma proteins. 1-D PAGE analysis of fractions depleted of dominant plasma proteins revealed treatment-specific changes in protein profiles. Protein bands that changed reproducibly in response to ER-alpha action were excised from the gel, separated by capillary LC, and identified by microspray ESI-MS. Using this method, the plasma levels of two proteins, transthyretin and apolipoprotein E, were shown to decrease in response to ER-alpha agonism. The method lacked the sensitivity to identify the known, 1000-fold less-abundant, estrogenic marker prolactin (PRL). However, using a commercial RIA and immunoblots, we showed that PRL levels increase significantly in response to treatment with the ER-alpha selective agonist, compound 1.