Development of an RNAi therapeutic for alpha-1-antitrypsin liver disease.

The autosomal codominant genetic disorder alpha-1 antitrypsin (AAT) deficiency (AATD) causes pulmonary and liver disease. Individuals homozygous for the mutant Z allele accumulate polymers of Z-AAT protein in hepatocytes, where AAT is primarily produced. This accumulation causes endoplasmic reticulum (ER) stress, oxidative stress, damage to mitochondria, and inflammation, leading to fibrosis, cirrhosis, and hepatocellular carcinoma. The magnitude of AAT reduction and duration of response from first-generation intravenously administered RNA interference (RNAi) therapeutic ARC-AAT and then with next-generation subcutaneously administered ARO-AAT were assessed by measuring AAT protein in serum of the PiZ transgenic mouse model and human volunteers. The impact of Z-AAT reduction by RNAi on liver disease phenotypes was evaluated in PiZ mice by measuring polymeric Z-AAT in the liver; expression of genes associated with fibrosis, autophagy, apoptosis, and redox regulation; inflammation; Z-AAT globule parameters; and tumor formation. Ultrastructure of the ER, mitochondria, and autophagosomes in hepatocytes was evaluated by electron microscopy. In mice, sustained RNAi treatment reduced hepatic Z-AAT polymer, restored ER and mitochondrial health, normalized expression of disease-associated genes, reduced inflammation, and prevented tumor formation. RNAi therapy holds promise for the treatment of patients with AATD-associated liver disease. ARO-AAT is currently in phase II/III clinical trials.

Protease-triggered siRNA delivery vehicles.

The safe and efficacious delivery of membrane impermeable therapeutics requires cytoplasmic access without the toxicity of nonspecific cytoplasmic membrane lysis. We have developed a mechanism for control of cytoplasmic release which utilizes endogenous proteases as a trigger and results in functional delivery of small interfering RNA (siRNA). The delivery approach is based on reversible inhibition of membrane disruptive polymers with protease-sensitive substrates. Proteolytic hydrolysis upon endocytosis restores the membrane destabilizing activity of the polymers thereby allowing cytoplasmic access of the co-delivered siRNA. Protease-sensitive polymer masking reagents derived from polyethylene glycol (PEG), which inhibit membrane interactions, and N-acetylgalactosamine, which targets asialoglycoprotein receptors on hepatocytes, were synthesized and used to formulate masked polymer-siRNA delivery vehicles. The size, charge and stability of the vehicles enable functional delivery of siRNA after subcutaneous administration and, with modification of the targeting ligand, have the potential for extrahepatic targeting.

Mating pair formation homologue TraG is a variable membrane protein essential for contact-independent type IV secretion of chromosomal DNA by Neisseria gonorrhoeae.

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the surrounding milieu. The DNA is effective in transforming gonococci in the population, and this mechanism of DNA donation may contribute to the high degree of genetic diversity in this species. Similar to other F-like T4SSs, the gonococcal T4SS requires a putative membrane protein, TraG, for DNA transfer. In F-plasmid and related systems, the homologous protein acts in pilus production, mating pair stabilization, and entry exclusion. We characterized the localization, membrane topology, and variation of TraG in N. gonorrhoeae. TraG was found to be an inner-membrane protein with one large periplasmic region and one large cytoplasmic region. Each gonococcal strain carried one of three different alleles of traG. Strains that carried the smallest allele of traG were found to lack the peptidoglycanase gene atlA but carried a peptidoglycan endopeptidase gene in place of atlA. The purified endopeptidase degraded gonococcal peptidoglycan in vitro, cutting the peptide cross-links. Although the other two traG alleles functioned for DNA secretion in strain MS11, the smallest traG did not support DNA secretion. Despite the requirement for a mating pair stabilization homologue, static coculture transformation experiments demonstrated that DNA transfer was nuclease sensitive and required active uptake by the recipient, thus demonstrating that transfer occurred by transformation and not conjugation. Together, these results demonstrate the TraG acts in a process of DNA export not specific to conjugation and that different forms of TraG affect what substrates can be transported.

Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection.

RNA interference (RNAi)-based therapeutics have the potential to treat chronic hepatitis B virus (HBV) infection in a fundamentally different manner than current therapies. Using RNAi, it is possible to knock down expression of viral RNAs including the pregenomic RNA from which the replicative intermediates are derived, thus reducing viral load, and the viral proteins that result in disease and impact the immune system's ability to eliminate the virus. We previously described the use of polymer-based Dynamic PolyConjugate (DPC) for the targeted delivery of siRNAs to hepatocytes. Here, we first show in proof-of-concept studies that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, we show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. These results suggest that coinjection of NAG-MLP and chol-siHBVs holds great promise as a new therapeutic for patients chronically infected with HBV.

Co-injection of a targeted, reversibly masked endosomolytic polymer dramatically improves the efficacy of cholesterol-conjugated small interfering RNAs in vivo.

Effective in vivo delivery of small interfering (siRNA) has been a major obstacle in the development of RNA interference therapeutics. One of the first attempts to overcome this obstacle utilized intravenous injection of cholesterol-conjugated siRNA (chol-siRNA). Although studies in mice revealed target gene knockdown in the liver, delivery was relatively inefficient, requiring 3 daily injections of 50 mg/kg of chol-siRNA to obtain measurable reduction in gene expression. Here we present a new delivery approach that increases the efficacy of the chol-siRNA over 500-fold and allows over 90% reduction in target gene expression in mice and, for the first time, high levels of gene knockdown in non-human primates. This improved efficacy is achieved by the co-injection of a hepatocyte-targeted and reversibly masked endosomolytic polymer. We show that knockdown is absolutely dependent on the presence of hepatocyte-targeting ligand on the polymer, the cognate hepatocyte receptor, and the cholesterol moiety of the siRNA. Importantly, we provide evidence that this increase in efficacy is not dependent on interactions between the chol-siRNA with the polymer prior to injection or in the bloodstream. The simplicity of the formulation and efficacy of this mode of siRNA delivery should prove beneficial in the use of siRNA as a therapeutic.

AtlA functions as a peptidoglycan lytic transglycosylase in the Neisseria gonorrhoeae type IV secretion system.

Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis.

In vivo gene expression analysis identifies genes required for enhanced colonization of the mouse urinary tract by uropathogenic Escherichia coli strain CFT073 dsdA.

Deletional inactivation of the gene encoding d-serine deaminase, dsdA, in uropathogenic Escherichia coli strain CFT073 results in a hypermotile strain with a hypercolonization phenotype in the bladder and kidneys of mice in a model of urinary tract infection (UTI). The in vivo gene expression profiles of CFT073 and CFT073 dsdA were compared by isolating RNA directly from the urine of mice challenged with each strain individually. Hybridization of cDNAs derived from these samples to CFT073-specific microarrays allowed identification of genes that were up- or down-regulated in the dsdA deletion strain during UTI. Up-regulated genes included the known d-serine-responsive gene dsdX, suggesting in vivo intracellular accumulation of d-serine by CFT073 dsdA. Genes encoding F1C fimbriae, both copies of P fimbriae, hemolysin, OmpF, a dipeptide transporter DppA, a heat shock chaperone IbpB, and clusters of open reading frames with unknown functions were also up-regulated. To determine the role of these genes as well as motility in the hypercolonization phenotype, mutants were constructed in the CFT073 dsdA background and tested in competition against the wild type in the murine model of UTI. Strains with deletions of one or both of the two P fimbrial operons, hlyA, fliC, ibpB, c0468, locus c3566 to c3568, or c2485 to c2490 colonized mouse bladders and kidneys at levels indistinguishable from wild type. CFT073 dsdA c2398 and CFT073 dsdA focA maintained a hypercolonization phenotype. A CFT073 dsdA dppA mutant was attenuated 10- to 50-fold in its colonization ability compared to CFT073. Our results support a role for d-serine catabolism and signaling in global virulence gene regulation of uropathogenic E. coli.

Natural transformation of Neisseria gonorrhoeae: from DNA donation to homologous recombination.

Gonococci undergo frequent and efficient natural transformation. Transformation occurs so often that the population structure is panmictic, with only one long-lived clone having been identified. This high degree of genetic exchange is likely necessary to generate antigenic diversity and allow the persistence of gonococcal infection within the human population. In addition to spreading different alleles of genes for surface markers and allowing avoidance of the immune response, transformation facilitates the spread of antibiotic resistance markers, a continuing problem for treatment of gonococcal infections. Transforming DNA is donated by neighbouring gonococci by two different mechanisms: autolysis or type IV secretion. All types of DNA are bound non-specifically to the cell surface. However, for DNA uptake, Neisseria gonorrhoeae recognizes only DNA containing a 10-base sequence (GCCGTCTGAA) present frequently in the chromosome of neisserial species. Type IV pilus components and several pilus-associated proteins are necessary for gonococcal DNA uptake. Incoming DNA is subject to restriction, making establishment of replicating plasmids difficult but not greatly affecting chromosomal transformation. Processing and integration of transforming DNA into the chromosome involves enzymes required for homologous recombination. Recent research on DNA donation mechanisms and extensive work on type IV pilus biogenesis and recombination proteins have greatly improved our understanding of natural transformation in N. gonorrhoeae. The completion of the gonococcal genome sequence has facilitated the identification of additional transformation genes and provides insight into previous investigations of gonococcal transformation. Here we review these recent developments and address the implications of natural transformation in the evolution and pathogenesis N. gonorrhoeae.

Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system.

The process of DNA donation for natural transformation of bacteria is poorly understood and has been assumed to involve bacterial cell death. Recently in Neisseria gonorrhoeae we found that mutations in three genes in the gonococcal genetic island (GGI) reduced the ability of a strain to act as a donor in transformation and to release DNA into the culture. To better characterize the GGI and the process of DNA donation, the 57 kb genetic island was cloned, sequenced and subjected to insertional mutagenesis. DNA sequencing revealed that the GGI has characteristics of a horizontally acquired genomic island and encodes homologues of type IV secretion system proteins. The GGI was found to be incorporated near the chromosomal replication terminus at the dif site, a sequence targeted by the site-specific recombinase XerCD. Using a plasmid carrying a small region of the GGI and the associated dif site, we demonstrated that this model island could be integrated at the dif site in strains not carrying the GGI and was spontaneously excised from that site. Also, we were able to delete the entire 57 kb region by transformation with DNA from a strain lacking the GGI. Thus the GGI was likely acquired and integrated into the gonococcal chromosome by site-specific recombination and may be lost by site-specific recombination or natural transformation. We made mutations in six putative type IV secretion system genes and assayed these strains for the ability to secrete DNA. Five of the mutations greatly reduced or completely eliminated DNA secretion. Our data indicate that N. gonorrhoeae secretes DNA via a specific process. Donated DNA may be used in natural transformation, contributing to antigenic variation and the spread of antibiotic resistance, and it may modulate the host immune response.