TitlePotent latency reversal by Tat RNA-containing nanoparticle enables multi-omic analysis of the HIV-1 reservoir
OverviewThe development of latency reversing agents that potently reactivate HIV
without inducing global T cell activation would benefit the field of HIV reservoir
research and could pave the way to a functional cure. Here, we explore the
reactivation capacity of a lipid nanoparticle containing TatmRNA (Tat-LNP) in
CD4 T cells from people living with HIV undergoing antiretroviral therapy
(ART). When combined with panobinostat, Tat-LNP induces latency reversal in
a significantly higher proportion of latently infected cells compared to PMA/
ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the
transcriptome of CD4 T cells, enabling the characterization of latently infected
cells in their near-native state. Upon latency reversal, we identify transcriptomic
differences between infected cells carrying an inducible provirus
and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic
differences at the protein level and provide evidence that the long
non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore,
p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation
of protein translation, suggesting that HIV-infected cells display distinct signatures
facilitating their long-term persistence. Tat-LNP represents a valuable
research tool for in vitro reservoir studies as it greatly facilitates the in-depth
characterization of HIV reservoir cells’ transcriptome and proteome profiles.
TitleEngineered tRNAs suppress nonsense mutations in cells and in vivo
OverviewNonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases1. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation2,3,4,5,6,7. However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP–sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression.
TitleTALENs delivered as mRNA cargo in LNPs cause LPA gene editing and Lp(a) reduction in vivo
Overview● Achieved proof-of-concept for TALEN-mediated Apo(a) reduction in vitro using TALEN mRNAs
● Demonstrated in vivo efficacy by LNP delivery of TALEN mRNA that led to Lp(a) reduction in a transgenic mouse model
● TALEN-mediated gene editing was dose responsive
● TALEN mRNA-LNP formulations were well-tolerated in vivo
● No detectable off-target activity in vitro
TitleLipid nanoparticle delivers phenylalanine ammonia lyase mRNA to the liver leading to catabolism and clearance of phenylalanine in a phenylketonuria mouse model
OverviewPhenylketonuria (PKU) is a genetic disorder affecting around 1 in 12,000 live births (1), caused by a mutation in the phenylalanine hydroxylase (PAH) gene in the liver which facilitates the catabolism of phenylalanine (Phe). Without a functional copy of PAH, levels of Phe in the blood and tissues rise, resulting in potentially life- threatening damage to the central nervous system. (2) Treatment options for PKU are limited, and center around adherence to a strict PKU diet that suffers from poor patient compliance. There are two approved drugs available, one of which must be used in conjunction with the PKU diet and another that has serious immuno- logical side effects. Here we demonstrate that the LUNAR® delivery technology is capable of delivering mRNA for a replacement enzyme, the bacterial phenylalanine ammonia lyase (avPAL), into the hepatic tissue of a PKU mouse, and that the enzyme is capable of metabolizing Phe and reducing serum levels of Phe for more than five days post-transfection. We further demonstrate the ability of LUNAR to deliver a plant-derived PAL protein with a similar impact on the level of serum Phe. Taken together these results demonstrate both the capability of LUNAR for the targeted delivery of PAL mRNA into hepatic tissue in vivo, replacing the defective PAH protein and successfully reducing serum Phe levels, thereby addressing the underlying cause of PKU symptoms. Secondly, that plant-based PAL proteins are a viable alternative to bacterial avPAL to reduce the immunogenic response.
TitleDownregulation of peripheral lipopolysaccharide binding protein impacts on perigonadal adipose tissue only in female mice
OverviewBackground and aims: The sexual dimorphism in fat-mass distribution and circulating leptin and insulin levels is well known, influencing the progression of obesity-associated metabolic disease. Here, we aimed to investigate the possible role of lipopolysaccharide-binding protein (LBP) in this sexual dimorphism. Methods: The relationship between plasma LBP and fat mass was evaluated in 145 subjects. The effects of Lbp downregulation, using lipid encapsulated unlocked nucleomonomer agent containing chemically modified siRNA delivery system, were evaluated in mice. Results: Plasma LBP levels were associated with fat mass and leptin levels in women with obesity, but not in men with obesity. In mice, plasma LBP downregulation led to reduced weight, fat mass and leptin gain after a high-fat and high-sucrose diet (HFHS) in females, in parallel to increased expression of adipogenic and thermogenic genes in visceral adipose tissue. This was not observed in males. Plasma LBP downregulation avoided the increase in serum LPS levels in HFHS-fed male and female mice. Serum LPS levels were positively correlated with body weight and fat mass gain, and negatively with markers of adipose tissue function only in female mice. The sexually dimorphic effects were replicated in mice with established obesity. Of note, LBP downregulation led to recovery of estrogen receptor alpha (Esr1) mRNA levels in females but not in males. Conclusion: LBP seems to exert a negative feedback on ERα-mediated estrogen action, impacting on genes involved in thermogenesis. The known decreased estrogen action and negative effects of metabolic endotoxemia may be targeted through LBP downregulation.
TitletRNA therapeutics burst onto startup scene
OverviewCompanies advance tRNA therapeutics to overcome mutant stoppages in protein synthesis shared by thousands of genetic diseases and cancers.
TitleDevelopment of an mRNA replacement therapy for phenylketonuria
OverviewPhenylketonuria (PKU) is an inborn error caused by deficiencies in phenylalanine (Phe) metabolism. Mutations in the phenylalanine hydroxylase (PAH) gene are the main cause of the disease whose signature hallmarks of toxically elevated levels of Phe accumulation in plasma and organs such as the brain, result in irreversible intellectual disability. Here, we present a unique approach to treating PKU deficiency by using an mRNA replacement therapy. A full-length mRNA encoding human PAH (hPAH) is encapsulated in our proprietary lipid nanoparticle LUNAR and delivered to a Pahenu2 mouse model that carries a missense mutation in the mouse PAH gene. Animals carrying this missense mutation develop hyperphenylalanemia and hypotyrosinemia in plasma, two clinical features commonly observed in the clinical presentation of PKU. We show that intravenous infusion of LUNAR-hPAH mRNA can generate high levels of hPAH protein in hepatocytes and restore the Phe metabolism in the Pahenu2 mouse model. Together, these data establish a proof of principle of a novel mRNA replacement therapy to treat PKU.
TitleSelective suppression of polyglutamine-expanded protein by lipid nanoparticle-delivered siRNA targeting CAG expansions in the mouse CNS
OverviewPolyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by expansion of cytosine-adenine-guanine (CAG)-trinucleotide repeats in causative genes. These diseases include spinal and bulbar muscular atrophy (SBMA), Huntington’s disease, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxias. Targeting expanded CAG repeats is a common therapeutic approach to polyQ diseases, but concomitant silencing of genes with normal CAG repeats may lead to toxicity. Previous studies have shown that CAG repeat-targeting small interfering RNA duplexes (CAG-siRNAs) have the potential to selectively suppress mutant proteins in in vitro cell models of polyQ diseases. However, in vivo application of these siRNAs has not yet been investigated. In this study, we demonstrate that an unlocked nucleic acid (UNA)- modified CAG-siRNA shows high selectivity for polyQ expanded androgen receptor (AR) inhibition in in vitro cell models and that lipid nanoparticle (LNP)-mediated delivery of the CAG-siRNA selectively suppresses mutant AR in the central nervous system of an SBMA mouse model. In addition, a subcutaneous injection of the LNP-delivered CAG-siRNA efficiently suppresses mutant AR in the skeletal muscle of the SBMA mouse model. These results support the therapeutic potential of LNP-delivered UNA-modified CAG-siRNAs for selective suppression of mutant proteins in SBMA and other polyQ diseases.
TitleCD8+ T cells mediate protection against Zika virus induced by an NS3-based vaccine
OverviewZika virus (ZIKV) is associated with congenital malformations in infants born to infected mothers, and with Guillain-Barré syndrome in infected adults. Development of ZIKV vaccines has focused predominantly on the induction of neutralizing antibodies, although a suboptimal antibody response may theoretically enhance disease severity through antibody-dependent enhancement (ADE). Here, we report induction of a protective anti-ZIKV CD8+ T cell response in the HLA-B*0702 Ifnar1-/- transgenic mice using an alphavirus-based replicon RNA vaccine expressing ZIKV nonstructural protein NS3, a potent T cell antigen. The NS3 vaccine did not induce a neutralizing antibody response but elicited polyfunctional CD8+ T cells that were necessary and sufficient for preventing death in lethally infected adult mice and fetal growth restriction in infected pregnant mice. These data identify CD8+ T cells as the major mediators of ZIKV NS3 vaccine-induced protection and suggest a new strategy to develop safe and effective anti-flavivirus vaccines.
TitleProperty Driven Design and Development of Lipids for Efficient Delivery of siRNA
OverviewIonizable cationic lipids are critical components involved in nanoparticle formulations, which are utilized in delivery platforms for RNA therapeutics. While general criteria regarding lipophilicity and measured pKa in formulation are understood to have impacts on utility in vivo, greater granularity with respect to the impacts of the structure on calculated and measured physicochemical parameters and the subsequent performance of those ionizable cationic lipids in in vivo studies would be beneficial.
TitleLipid Nanoparticle Formulation Increases Efficiency of DNA-Vectored Vaccines/Immunoprophylaxis in Animals Including Transchromosomic Bovines
OverviewThe use of nucleic acid as a drug substance for vaccines and other gene-based medicines continues to evolve. Here, we have used a technology originally developed for mRNA in vivo delivery to enhance the immunogenicity of DNA vaccines. We demonstrate that neutralizing antibodies produced in rabbits and nonhuman primates injected with lipid nanoparticle (LNP)-formulated Andes virus or Zika virus DNA vaccines are elevated over unformulated vaccine.
TitleAnti-HFRS Human IgG Produced in Transchromosomic Bovines Has Potent Hantavirus Neutralizing Activity and Is Protective in Animal Models
OverviewWe explored an emerging technology to produce anti-Hantaan virus (HTNV) and anti-Puumala virus (PUUV) neutralizing antibodies for use as pre- or post-exposure prophylactics.
TitleChallenges and Potential Solutions for Development of Successful Potency Assay in mRNA Therapeutics
OverviewmRNA Therapeutics require development of potency assays early during the pre-clinical stage. Conventional potency methods for protein biologic may not apply directly to mRNA therapeutic drug substance. Cell-based potency assays have to be carefully evaluated to make sure that the read-outs correspond to the actual potency of the drug substance and not an artifactual value. mRNAs can be evaluated for potency at level of protein expression (cell-free), cellular protein expression/stability and enzyme activity in vitro.
TitleSystemic delivery of factor IX messenger RNA for protein replacement therapy
OverviewSafe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B.