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Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel in vivodelivery approach based on an engineered transposase, Sleeping Beauty(SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene. This combinatorial approach achieved correction of ornithine transcarbamylase deficiency in the neonatal Spfashmouse model following a single delivery to dividing hepatocytes in the newborn liver. Correction remained stable into adulthood, while a conventional rAAV approach resulted in a return to the disease state. In non-human primates, integration by transposition, mediated by this technology, improved gene expression 10-fold over conventional rAAV-mediated gene transfer while requiring 5-fold less vector. Additionally, integration site analysis confirmed a random profile while specifically targeting TA dinucleotides across the genome. Together, these findings demonstrate that transposable elements can improve rAAV-delivered therapies by lowering the vector dose requirement and associated toxicity while expanding target cell types.

Genomic medicines have significant therapeutic potential to cure rare genetic diseases and cancer, but currently, most treatments rely on ex vivocell editing, which is time consuming, costly, and suffers from logistic and manufacturing challenges. In contrast, in vivodelivery of genomic medicines would unlock new therapeutic areas and provide broader patient access. However, the success of in vivodelivery is dependent on the development of safe delivery vehicles capable of transporting the genomic medicines to their intended target tissues and cells. In recent years, lipid nanoparticles (LNPs) have emerged as versatile delivery solutions for RNA therapeutics but have seldom been used fo r in vivodelivery beyond liver and muscle tissue. Challenges in extrahepatic delivery of LNPs include adequate tissue-specific targeting to achieve therapeutic levels of genome editing while avoiding anti-drug immune responses. We have developed novel LNPs designed to deliver RNA payloads to either T cells or hematopoietic stem cells (HSCs) in vivo.

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 vivoactivity. 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 vivogenome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.

Small interfering RNAs (siRNAs) direct Argonaute proteins, the core components of the RNA-induced silencing complex (RISC), to cleave complementary target RNAs. Here, the authors describe a method to purify active RISC containing a single, unique small RNA guide sequence. They begin by capturing RISC using a complementary 2′-O-methyl oligonucleotide tethered to beads. Unlike other methods that capture RISC but do not allow its recovery, their strategy purifies active, soluble RISC in good yield. They use this strategy to purify fly Ago1- and Ago2-RISC, as well as mouse AGO2-RISC. The method can discriminate among RISCs programmed with different guide strands, making it possible to deplete and recover specific RISC populations. Endogenous microRNA:Argonaute complexes can also be purified from cell lysates. The method scales readily and takes less than a day to complete.

Small RNAs loaded into Argonaute proteins direct silencing of complementary target mRNAs. It has been proposed that multiple, imperfectly complementary small interfering RNAs or microRNAs, when bound to the 3' untranslated region of a target mRNA, function cooperatively to silence target expression. We report that, in cultured human HeLa cells and mouse embryonic fibroblasts, Argonaute1 (Ago1), Ago3, and Ago4 act cooperatively to silence both perfectly and partially complementary target RNAs bearing multiple small RNA-binding sites. Our data suggest that for Ago1, Ago3, and Ago4, multiple, adjacent small RNA-binding sites facilitate cooperative interactions that stabilize Argonaute binding. In contrast, small RNAs bound to Ago2 and pairing perfectly to an mRNA target act independently to silence expression. Noncooperative silencing by Ago2 does not require the endoribonuclease activity of the protein: A mutant Ago2 that cannot cleave its mRNA target also silences noncooperatively. We propose that Ago2 binds its targets by a mechanism fundamentally distinct from that used by the three other mammalian Argonaute proteins.

RNA interference (RNAi) has been established as an important tool for functional genomics studies and has great promise as a therapeutic intervention for human diseases. In mammalian cells, RNAi is conventionally induced by 19–27-bp RNA duplexes generated by hybridization of two complementary oligonucleotide strands (oligos). Here we describe a novel class of RNAi molecules composed of a single 25–28-nucleotide (nt) oligo. The oligo has a 16-nt mRNA targeting region, followed by an additional 8–10 nt to enable self-dimerization into a partially complementary duplex. Analysis of numerous diverse structures demonstrates that molecules composed of two short helices separated by a loop can efficiently enter and activate the RNA-induced silencing complex (RISC). This finding enables the design of highly effective single-oligo compounds for any mRNA target.

Echoencephalograms were obtained for 118 of 121 successive infants who were admitted to the Stanford intensive care nursery, weighed 1,000 g or less at birth, and survived long enough for at least one study to be performed. Eightyeight of these infants survived and were followed up for 1 to 3 years; psychometric testing (Bayley Scales of Infant Development, Stanford-Binet Intelligence Scale, or both) was performed on 81% of these infants. Subependymal-intraventricular hemorrhages or intraparenchymal hemorrhages were associated with impaired development, but ventriculomegaly was not. The absence of echoencephalographic abnormalities did not exclude the possibility of impaired development in many infants. Periventricular leukomalacia was not observed. These data support independent scoring of subependymal intraventricular hemorrhages, intraparenchymal hemorrhages, and ventriculomegaly, rather than use of combined scales for prognostic purposes.

Total bilirubin formation (TBF) in the rat after a short period of common bile duct ligation was studied by measuring the pulmonary excretion rate of CO (VECO). At postoperative day 3, the VECOof experimental animals was higher when compared with the pre‐operative VECO(p < 0.005); whereas the VECOof control animals did not change. Also, on the 3rd postoperative day, the relative rate of early labeling of bilirubin following the administration of δ‐aminolevulinic acid‐5‐14C, a preferential hepatic heme synthesis precursor, was similar between the experimental and control animals; only the experimental animals had an abnormal peroxide hemolysis test. We conclude that common bile duct ligation in the rat is associated with elevations in the VECO, indicating significant increases in TBF, and the source of the increase is probably of erythropoietic origin. This finding may be relevant to the understanding of the pathophysiology of obstructive jaundice in human neonates.

The rate of total bilirubin production and the concomitant relative rate of early-labeled bilirubin (ELB) formation were studied in adult Wistar rats with short-term common bile duct ligation. The pulmonary excretion rate of CO (Veco), an index of total bilirubin production, was measured preoperatively and postoperatively on day 3 and on day 5 or 6. While the Vecomeasurements of control and experimental animals were similar preoperatively, the postoperative Vecomeasurements of the experimental animals averaged 30% higher than those of control animals. On postoperative day 3, the relative rate of ELB formation was measured by the recovery of excreted 14CO after the administration of 14C-labeled 8-aminolevulinic acid (ALA) or glycine as nonerythropoietic or total heme synthesis precursor, respectively. No significant differences were found in the 14CO recovered from either compound during the study period between the experimental and control animals. The half-life of 51Cr-labeled transfused red blood cells was decreased by nearly 50% in bile duct ligated rats compared to that in the sham operated ones. We conclude that hemolysis is a major factor contributing to the increase in total bilirubin production even after short-term biliary obstruction in the rat.

Total bilirubin formation (TBF) in the rat after a short period of common bile duct ligation was studied by measuring the pulmonary excretion rate of CO (VECO). At postoperative day 3, the VECOof experimental animals was higher when compared with the pre-operative VECO(p < 0.005); whereas the VECOof control animals did not change. Also, on the 3rd postoperative day, the relative rate of early labeling of bilirubin following the administration of δ-aminolevulinic acid-5-14C, a preferential hepatic heme synthesis precursor, was similar between the experimental and control animals; only the experimental animals had an abnormal peroxide hemolysis test. We conclude that common bile duct ligation in the rat is associated with elevations in the VECO, indicating significant increases in TBF, and the source of the increase is probably of erythropoietic origin. This finding may be relevant to the understanding of the pathophysiology of obstructive jaundice in human neonates.

The rate of total bilirubin production and the concomitant relative rate of early-labeled bilirubin (ELB) formation were studied in adult Wistar rats with short-term common bile duct ligation. The pulmonary excretion rate of CO (Veco), an index of total bilirubin production, was measured preoperatively and postoperatively on day 3 and on day 5 or 6. While the Vecomeasurements of control and experimental animals were similar preoperatively, the postoperative Vecomeasurements of the experimental animals averaged 30 higher than those of control animals. On postoperative day 3, the relative rate of ELB formation was measured by the recovery of excreted 14CO after the administration of 14C-labeled 8-aminolevulinic acid (ALA) or glycine as nonerythropoietic or total heme synthesis precursor, respectively. No significant differences were found in the 14CO recovered from either compound during the study period between the experimental and control animals. The half-life of 51Cr-labeled transfused red blood cells was decreased by nearly 50 in bile duct ligated rats compared to that in the sham operated ones. We conclude that hemolysis is a major factor contributing to the increase in total bilirubin production even after short-term biliary obstruction in the rat.

Common bile duct ligation in the adult male Wistar rat increases the rate of total bilirubin formation (TBF) as estimated by the excretion of carbon monoxide (VeCO). This increase is not due to elevation in the hepatic subcomponent of “earlylabeled” bilirubin (ELB) formation as measured by 14C-4-Δ-aminolevulinic (J Pediatr Gastroenterol Nutr 3:790, 1984). This study examines total ELB formation in rats 3 days after complete common bile duct ligation. VeCO was measured preoperatively and on the third day after creation of complete biliary obstruction. The rate of ELB formation was measured by recovery of exhaled 14CO following intraperitoneal injection of 14C-2-glycine (20 μCi/100gm) on the third postoperative day. Preoperatively, there was no significant difference in the VeCO between control and experimental rats. On the third postoperative day, the VeCO of control rats had returned to baseline from an elevated rate, due to the surgery, while the VeCO of experimental rats was increased by 31% (paired t-test, p<0.02). Total fractional recovery of injected label over a 36 hr collection period was similar for control (8.5±0.7 × 10−5, n=8) and experimental (8.2±0.2 ± 10−5, n=7) rats. Based on these data, we conclude that the rise in TBF in this obstructive model is not due to ELB formation. The results further suggest that increased red cell destruction occurs rapidly after short-term biliary obstruction, and may cause the increased TBF, contributing to the pathogenesis of obstructive jaundice.

All infants under 1001 gms were studied prospectively with ECHO within 24 hrs of admission to the Stanford ICN between 1/1/80 and 6/30/83. They were re-evaluated at least twice weekly, and scored by Papille's classification for ICH and for atrophy and hydrocephalus (HC). Of the 150 infants, 92 (61%) were discharged and examined yearly. Seven were excluded (4 deaths, 1 encephalocele, 1 lost to followup, 1 late non-neonatal neurologic injury). (± SD)These data suggest that although infants with Grade I & II ICH have more abnormal neurodevelopmental (ND) exams, these infants still have good potential for normal neurological function (74%). However, infants with Grade III & IV ICH or isolated atrophy and/or HC may have a poorer prognosis, with no normal exams. Infants with a normal ECHO but an abnormal exam had low birth weights when compared to those with a normal ND exam and also had serious complications of Candida sepsis (3), asphyxia (1), hypothyroidism (1), pneumonia (1), and histiocytosis X (1).