Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.

A phase I dose-escalation study of PEP02 (irinotecan liposome injection) in combination with 5-fluorouracil and leucovorin in advanced solid tumors.

BACKGROUND: PEP02 (also known as MM-398, nal-IRI) is a novel nanoparticle formulation of irinotecan encapsulated in liposomes. The aims of this study were to investigate the dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and pharmacokinetics (PK) of PEP02 in combination with 5-FU and LV, in patients with advanced refractory solid tumors. METHODS: Patients were enrolled in cohorts to receive PEP02 from 60 to 120 mg/m2 (dose expressed as the irinotecan hydrochloride trihydrate salt) as a 90-min intravenous infusion on day 1, followed by 24 h infusion of 5-FU 2,000 mg/m2 and LV 200 mg/m2 on days 1 and 8, every 3 weeks. RESULTS: A total of 16 patients were assigned to four dose levels, 60 (three patients), 80 (six patients), 100 (five patients) and 120 mg/m2 (two patients). DLT was observed in four patients, two at the 100 mg/m2 dose level (one had grade III infection with hypotension and grade III hemorrhage; the other had grade III diarrhea and grade IV neutropenia), and two at the 120 mg/m2 dose level (one had grade III diarrhea and grade IV neutropenia; the other had grade III diarrhea). The MTD of PEP02 was determined as 80 mg/m2. The most common treatment-related adverse events were nausea (81%), diarrhea (75%) and vomiting (69%). Among the six patients who received the MTD, one patient exhibited partial response, four patients had stable disease and one showed progressive disease. Pharmacokinetic data showed that PEP02 had a lower peak plasma concentration, longer half-life, and increased area under the plasma concentration-time curve from zero to time t of SN-38 than irinotecan at similar dose level. CONCLUSIONS: The MTD of PEP02 on day 1 in combination with 24-h infusion of 5-FU and LV on days 1 and 8, every 3 weeks was 80 mg/m2, which will be the recommended dose for future studies. TRIAL REGISTRATION: The trial was retrospectively registered ( NCT02884128 ) with date of registration: August 12, 2016.

PEPCOL: a GERCOR randomized phase II study of nanoliposomal irinotecan PEP02 (MM-398) or irinotecan with leucovorin/5-fluorouracil as second-line therapy in metastatic colorectal cancer.

A multicenter, open-label, noncomparative, randomized phase II study (PEPCOL) was conducted to evaluate the efficacy and safety of the irinotecan or PEP02 (MM-398, nanoliposomal irinotecan) with leucovorin (LV)/5-fluorouracil (5-FU) combination as second-line treatment in patients with metastatic colorectal cancer (mCRC). Patients with unresectable mCRC who had failed one prior oxaliplatin-based first-line therapy were randomized toirinotecan with LV/5-FU (FOLFIRI) or PEP02 with LV/5-FU (FUPEP; PEP02 80 mg/m(2) with LV 400 mg/m(2) on day 1 and 5-FU 2400 mg/m(2) on days 1-2). Bevacizumab (5 mg/kg, biweekly) was allowed in both arms. The primary endpoint was 2-month response rate (RR). Fifty-five patients were randomized (FOLFIRI, n = 27; FUPEP, n = 28). In the intent-to-treat population (n = 55), 2-month RR response rate was observed in two (7.4%) and three (10.7%) patients in the FOLFIRI and FUPEP arms, respectively. The most common grade 3-4 adverse events reported in the respective FOLFIRI and FUPEP arms were diarrhea (33% vs. 21%), neutropenia (30% vs. 11%), mucositis (11% vs. 11%), and grade 2 alopecia (26% vs. 25%). FUPEP has activity and acceptable safety profile in oxaliplatin-pretreated mCRC patients.

A soluble chimeric inhibitor of C3 and C5 convertases, complement activation blocker-2, prolongs graft survival in pig-to-rhesus monkey heart transplantation.

Complement plays a critical role in many pathologic processes and in xenograft rejection. Therefore, effective complement inhibitors are of great interest. In pig-to-primate organ transplantation, hyperacute rejection results from antibody deposition and complement activation. Complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF) and membrane cofactor protein, inhibits C3 and C5 convertases of both classical and alternative pathways. CAB-2 reduces complement-mediated tissue injury of a pig heart perfused ex vivo with human blood. Therefore, we studied the efficacy of CAB-2 when a pig heart is transplanted heterotopically into rhesus monkeys receiving no immunosuppression. Graft survival in three control monkeys was 1.26 +/- 0.2 h; it was markedly prolonged in eight monkeys that received CAB-2. Of the six monkeys that received a single dose of CAB-2 (15 mg/kg i.v.), four had graft survivals of 21, 95, 96, and 108 h, and two died at 7 to 11 h post-transplant with a beating graft, as a result of technical complications. The two monkeys given multiple doses of CAB-2 had graft survivals of 95 and 96 h. CAB-2 markedly inhibited complement activation, as shown by a strong reduction in generation of C3a and SC5b-9. At graft rejection, tissue deposition of iC3b, C4 and C9 was similar or slightly reduced from controls, and deposition of IgG, IgM, C1q and fibrin did not change. Thus, complement inhibition with CAB-2 abrogates hyperacute rejection of pig hearts transplanted into rhesus monkeys, but does not prevent delayed/acute vascular rejection. These studies demonstrate that the beneficial effects of complement inhibition on survival of a pig heart xenograft in rhesus monkeys are similar to those in other primate species and that CAB-2 may be useful in xenotransplantation and other complement-mediated conditions.