1 Abstract Adoptive cell therapy using tumor antigen-targeting T cell receptors (TCRs) offers a compelling approach to treat both hematological cancers and solid tumors due to broad antigen accessibility and the ability to target cancer-specific neoantigens. However, unlike clinically validated second generation CAR-T cells bearing built-in co-stimulatory signaling modules (i.e. 41BB or CD28), TCR-T cells receive little to no co-stimulation within most tumor microenvironments leading to attenuated cellular responses. Additionally, CD4+ TCR-T cells engineered to express HLA-Class I restricted TCRs possess minimal T-helper cell activity and thus do not effectively mobilize CD8+ TCR-T cells or host anti-tumor immune responses. To address these limitations, we used CRISPR-Cas9 to engineer TCR-T cells with targeted integration of chimeric CD8 constructs containing intracellular co-stimulatory domains. We found that expression of wild-type CD8αβ, but not CD8αα, could promote CD4+ T cell activities in HLA-Class I restricted TCR-T cells. However, this was insufficient to drive durable anti-tumor responses in challenging tumor mouse models when using a high-affinity WT1-directed TCR. To address this, several CD8 co-stimulatory fusion constructs containing CD28 or 41BB intracellular domains were designed and screened, identifying two CD8-41BB based chimeras that substantially increased TCR-T cell activity relative to wild-type CD8αβ. WT1-TCR-T cells co-expressing the CD8-41BB fusions demonstrated not only enhanced CD4+ activity including strong and polarized Th1-type cytokine secretion, but also enhanced the proliferation, cytokine release, and cytotoxicity of CD8+ CTLs. Remarkably, when combined with TGFBR2 gene disruption, WT1-TCR-T cells co-expressing CD8-41BB receptors were able to completely regress established cell line-derived ovarian tumors, showed robust in vivo expansion and persistence, and provided long-term protection from tumor rechallenge. Importantly, the specificity profile of the WT1-TCR including its HLA-A*02:01 restriction and WT1 peptide recognition motif was preserved upon expression of CD8-41BB. To simplify cell engineering processes for clinical applications, we configured a homology directed repair (HDR) cassette to allow for efficient CRISPR-Cas9-based insertion of both the TCR and CD8-41BB transgenes in the TRAC locus in a single step with >80% efficiency. Lastly, the enhanced activity conferred by CD8-41BB expression was validated with a second clinically relevant TCR targeting PRAME, suggesting this platform can be a universal approach for enhancing the therapeutic potential of TCR-based cell therapies.

“It is amazing to witness the speed by which discovery can now be translated into practice.”Abi Pinchbeck (Editor, BioInsights) speaks to Laura Sepp-Lorenzino (Scientific Advisor and former CSO, Intellia Therapeutics) about the evolution and future of gene editing, focusing on CRISPR-based therapies. They also discuss major scientific advancements, clinical successes, delivery challenges, and the promise of emerging technologies, such as base editing, prime editing, and DNA writing.

Rare diseases are serious and often chronic conditions that affect a small number of individuals. However, with over 7,000 rare diseases identified, their cumulative global numbers and impact are substantial. A considerable proportion of these conditions is caused by genetic abnormalities. Among these, monogenic disorders are of particular relevance, as they are caused by mutations in specific genes. The development of gene therapy, and more specifically, gene editing, offers innovative approaches to treat these rare diseases. A significant challenge associated with the implementation of such strategies concerns the delivery of gene editing tools. Nonviral vectors based on nanomaterials have demonstrated considerable potential as promising alternatives to viral vectors, thereby overcoming their disadvantages. The biocompatibility and tunability of nanoparticles, along with their potential capacity to target diverse tissues, positions them as a promising therapeutic approach for the treatment of a wide range of organ-specific rare diseases. Here, we review current progress in the development and evaluation of novel nanomedicine strategies for gene editing in rare diseases, highlighting new gene editing approaches, delivery systems, and potential targets.

BACKGROUND Hereditary angioedema is a rare genetic disease that leads to severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy based on clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1), with the goal of lifelong control of angioedema attacks after a single dose. METHODS In this phase 1 dose-escalation portion of a combined phase 1-2 trial of NTLA-2002 in adults with hereditary angioedema, we administered NTLA-2002 at a single dose of 25 mg, 50 mg, or 75 mg. The primary end points were the safety and side-effect profile of NTLA-2002 therapy. Secondary and exploratory end points included pharmacokinetics, pharmacodynamics, and clinical efficacy determined on the basis of investigator-confirmed angioedema attacks. RESULTS Three patients received 25 mg of NTLA-2002, four received 50 mg, and three received 75 mg. At all dose levels, the most common adverse events were infusion-related reactions and fatigue. No dose-limiting toxic effects, serious adverse events, grade 3 or higher adverse events, or clinically important laboratory findings were observed after the administration of NTLA-2002. Dose-dependent reductions in the total plasma kallikrein protein level were observed between baseline and the latest assessment, with a mean percentage change of -67% in the 25-mg group, -84% in the 50-mg group, and -95% in the 75-mg group. The mean percentage change in the number of angioedema attacks per month between baseline and weeks 1 through 16 (primary observation period) was -91% in the 25-mg group, -97% in the 50-mg group, and -80% in the 75-mg group. Among all the patients, the mean percentage change in the number of angioedema attacks per month from baseline through the latest assessment was -95%. CONCLUSIONS In this small study, a single dose of NTLA-2002 led to robust, dose-dependent, and durable reductions in total plasma kallikrein levels, and no severe adverse events were observed. In exploratory analyses, reductions in the number of angioedema attacks per month were observed at all dose levels. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830.)

Nucleic Acid TherapeuticsVol. 33, No. 4 AnnouncementFree AccessRosalind Franklin Society Proudly Announces the 2022 Award Recipient for Nucleic Acid TherapeuticsLaura Sepp-LorenzinoLaura Sepp-LorenzinoIntellia Therapeutics, Boston, MA, USASearch for more papers by this authorPublished Online:9 Aug 2023https://doi.org/10.1089/nat.2023.29007.rfs2022AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail The Rosalind Franklin Society (RFS), in partnership with Mary Ann Liebert, Inc., publishers, enthusiastically congratulate our distinguished recipient of the 2022 annualRFS Award in Sciencefor this journal, which recognizes the outstanding research and published work of women and underrepresented minority scientists, physicians, and engineers.Dr. Laura Sepp-Lorenzino is an incredible woman from Argentina, and has helped the field a tremendous amount. She has served as a great role model for women and minorities on the pharma/biotech area in the field, and has served Nucleic Acid Therapeutics as a reviewer, author, and editorial board member.BiosketchLaura Sepp-Lorenzino, PhD, is chief scientific officer and executive vice president at Intellia Therapeutics, a leading clinical-stage genome editing company developing novel, potentially curative medicines leveraging CRISPR-based technologies. In addition to her extensive experience in nucleic acid therapies, Sepp-Lorenzino has expertise in oncology drug discovery and development acquired earlier in her career by leading the Cancer Research Department at Merck West Point and working as an assistant attending molecular biologist at Memorial Sloan Kettering Cancer Center. She received her professional degree in biochemistry from the University of Buenos Aires, Argentina, and both her MS and PhD in biochemistry from New York University. Sepp-Lorenzino is a member of the board of directors of Taysha Gene Therapies, the Alliance for Regenerative Medicine, and the Oligonucleotide Therapeutics Society and sits on the scientific advisory boards of Thermo Fisher Scientific, the UK Nucleic Acid Therapy Accelerator, and Arsenal Capital Partners. She is a member of the editorial board of Nucleic Acid Therapeutics.FiguresReferencesRelatedDetails Volume 33Issue 4Aug 2023 InformationCopyright 2023, Mary Ann Liebert, Inc., publishersTo cite this article:Laura Sepp-Lorenzino.Rosalind Franklin Society Proudly Announces the 2022 Award Recipient for Nucleic Acid Therapeutics.Nucleic Acid Therapeutics.Aug 2023.233-233.http://doi.org/10.1089/nat.2023.29007.rfs2022Published in Volume: 33 Issue 4: August 9, 2023PDF download

Downregulation of genes involved in the secondary pathology of Duchenne muscular dystrophy, for example, inflammation, fibrosis, and adiposis, is an interesting approach to ameliorate degeneration of muscle and replacement by fibrotic and adiposis tissue. Small interfering RNAs (siRNAs) are able to downregulate target genes, however, delivery of siRNAs to skeletal muscle still remains a challenge. We investigated delivery of fully chemically modified, cholesterol-conjugated siRNAs targeting Alk4, a nontherapeutic target that is expressed highly in muscle. We observed that a single intravenous or intraperitoneal (IP) injection of 10 mg/kg resulted in significant downregulation of Alk4 mRNA expression in skeletal muscles in both wild-type and mdx mice. Treatment with multiple IP injections of 10 mg/kg led to an overall reduction of Alk4 expression, reaching significance in tibialis anterior (39.7% ± 6.2%), diaphragm (32.7% ± 5.8%), and liver (41.3% ± 29.9%) in mdx mice. Doubling of the siRNA dose did not further increase mRNA silencing in muscles of mdx mice. The chemically modified conjugated siRNAs used in this study are very promising for delivery to both nondystrophic and dystrophic muscles and could have major implications for treatment of muscular dystrophy pathology.

Supplementary Table S1 from Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling

Background & Aims: Current therapy for chronic hepatitis B virus (cHBV) infection involves lifelong treatment. New treatments that enable HBV functional cure would represent a clinically meaningful advance. ALN-HBV and VIR-2218 are investigational RNA interference therapeutics that target all major HBV transcripts. Methods: We report on: i) the safety of single doses of VIR-2218 (modified from ALN-HBV by enhanced stabilization chemistry plus technology to reduce off-target, seed-mediated binding while maintaining on-target antiviral activity) and ALN-HBV in hu-manized mice; ii) a cross-study comparison of the safety of single doses of VIR-2218 and ALN-HBV in healthy human volunteers (n = 24 and n = 49, respectively); and iii) the antiviral activity of two doses of 20, 50, 100, 200 mg of VIR-2218 (total n = 24) vs. placebo (n = 8), given 4 weeks apart, in participants with cHBV infection. Results: In humanized mice, alanine aminotransferase (ALT) levels were markedly lower following administration of VIR-2218 compared with ALN-HBV. In healthy volunteers, post-treatment ALT elevations occurred in 28% of participants receiving ALN-HBV compared with none in those receiving VIR-2218. In participants with cHBV infection, VIR-2218 was associated with dose-dependent reductions in hepatitis B surface antigen (HBsAg). The greatest mean reduction of HBsAg at Week 20 in par-ticipants receiving 200 mg was 1.65 log IU/ml. The HBsAg reduction was maintained at 0.87 log IU/ml at Week 48. No participants had serum HBsAg loss or hepatitis B surface antibody seroconversion. Conclusions: VIR-2218 demonstrated an encouraging hepatic safety profile in preclinical and clinical studies as well as dose -dependent HBsAg reductions in patients with cHBV infection. These data support future studies with VIR-2218 as part of combination regimens with a goal of HBV functional cure. Trial registration: ClinicalTrials.gov identifiers: NCT02826018 and NCT03672188. (c) 2023 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Background and aims: Waning SARS-CoV-2 specific antibody (Ab) responses and high infection rates has led to the widespread use of 3rd/4th vaccine doses (V3/V4) in vulnerable patients.However, the immune response to V4 in patients with suboptimal prior vaccine responses is unexplored.We recruited liver transplant recipients (LT) and autoimmune hepatitis patients (AIH) to assess the immune responses to V4, including to SARS-CoV-2 omicron (B.1.1.529)variant.Method: Anti-spike (S) and anti-nucleocapsid (N) IgG titres were assessed (Roche assay) after V4 (24 Pfizer BNT162b2, 8 Moderna mRNA-1273) in 32 immunosuppressed patients, including 23 with liver disease (15 LT, 8 AIH) and 9 inflammatory bowel disease (IBD) patients.All patients had absent (13/32 (41%); 11 LT, 2 AIH) or low Ab responses (19/32 (59%); 4 LT, 6 AIH, 9 IBD) after 2 vaccines (V2) defined as anti-S IgG level <400 AU/ml.Anti-S and anti-N IgG titres performed after V2/V3.WT and omicron specific T cell responses were assessed by IFN-γ ELISpot assay in 30/32 patients using peptide pools covering whole-S and minipools of S-specific mutated peptides.Results: There were significant increases in anti-S IgG titres following V3 in all groups, stratified by those with absent and low anti-S Abs post V2 (Fig. 1A).All patients with a low post V2 response had a significant increase in anti-S Ab responses after V3 that was sustained (but not further enhanced) after V4.In liver patients with no anti-S Ab responses after V2, 7/13 (54%) developed anti-S Abs after V3 (0.41 AU/ml vs. 126.72AU/ml; p = 0.016), and importantly these responses were further significantly enhanced following V4 (126.72 AU/ml vs. 3212.90AU/ml; p = 0.002).However, 3/13 (23%) with absent anti-S Abs after V2 remained nonresponsive after V3 and V4.T cell responses to full-S WT and omicron peptides were heterogenous, but generally of high magnitude and detectable in 29/30 patients (Fig. 1B).There was a loss against Omicron (51.8%) when T cell responses were assessed using the variant mini pools (Fig. 1B).LT had significantly reduced response compared to IBD against omicron (mean 796.3 SFC/10 6 vs. 1589 SFC/10 6 ; p = 0.0294) and WT (mean 897.6 SFC/10 6 vs. 1817 SFC/10 6 ; p = 0.0274) (Fig. 1B).Importantly, the 3 seronegative patients after V4, mounted a robust T cell response to WT (mean 882.5 SFC/10 6 ) and omicron (mean 2280 SFC/10 6 ).There was no correlation between humoral and cellular responses (R 2 = 0.01). Conclusion:In liver and IBD patients, low anti-S Ab responses after 2 COVID19 vaccines may be enhanced by V3 and sustained by V4.Patients with absent anti-S Ab responses after 2 doses may have a significant increase in anti-S Ab titres after both V3 and V4.However, a small subset remain non-responsive and should be prioritised for prophylactic strategies.Importantly, immunosuppressed liver patients develop robust T cell responses to WT and omicron independent of anti-S Ab titres.

Abstract Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc–siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2′–5′-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.

RNA interference (RNAi) offers the potential to treat disease at the earliest onset by selectively turning off the expression of target genes, such as intracellular oncogenes that drive cancer growth. However, the development of RNAi therapeutics as anti-cancer drugs has been limited by both a lack of efficient and target cell-specific delivery systems and the necessity to overcome numerous intracellular barriers, including serum/ lysosomal instability, cell membrane impermeability, and limited endosomal escape. Here, we combine two technologies to achieve posttranscriptional gene silencing in tumor cells: Centyrins, alternative scaffold proteins binding plasma membrane receptors for targeted delivery, and small interfering RNAs (siRNAs), chemically modified for high metabolic stability and potency. An EGFR Centyrin known to internalize in EGFR-positive tumor cells was site-specifically conjugated to a beta-catenin (CTNNb1) siRNA and found to drive potent and specific target knockdown by free uptake in cell culture and in mice inoculated with A431 tumor xenografts (EGFR amplified). The generalizability of this approach was further demonstrated with Centyrins targeting multiple receptors (e.g., BCMA, PSMA, and EpCAM) and siRNAs targeting multiple genes (e.g., CD68, KLKb1, and SSB1). Moreover, by installing multiple conjugation handles, two different siRNAs were fused to a single Centyrin, and the conjugate was shown to simultaneously silence two different targets. Finally, by specifically pairing EpCAM-binding Centyrins that exhibited optimized internalization profiles, we present data showing that an EpCAM Centyrin CTNNb1 siRNA conjugate suppressed tumor cell growth of a colorectal cancer cell line containing an APC mutation but not cells with normal CTNNb1 signaling. Overall, these data demonstrate the potential of CentyrinsiRNA conjugates to target cancer cells and silence oncogenes, paving the way to a new class of anticancer drugs.

BACKGROUND & AIMS:Hepatitis B virus (HBV) infection persists because the virus-specific immune response is dysfunctional. Therapeutic vaccines might be used to end immune tolerance to the virus in patients with chronic infection, but these have not been effective in patients so far. In patients with chronic HBV infection, high levels of virus antigens might prevent induction of HBV-specific immune responses. We investigated whether knocking down expression levels of HBV antigens in liver might increase the efficacy of HBV vaccines in mice. METHODS:We performed studies with male C57BL/6 mice that persistently replicate HBV (genotype D, serotype ayw)-either from a transgene or after infection with an adeno-associated virus that transferred an overlength HBV genome-and expressed HB surface antigen at levels relevant to patients. Small hairpin or small interfering (si)RNAs against the common 3'-end of all HBV transcripts were used to knock down antigen expression in mouse hepatocytes. siRNAs were chemically stabilized and conjugated to N-acetylgalactosamine to increase liver uptake. Control mice were given either entecavir or non-HBV-specific siRNAs and vaccine components. Eight to 12 weeks later, mice were immunized twice with a mixture of adjuvanted HBV S and core antigen, followed by a modified Vaccinia virus Ankara vector to induce HBV-specific B- and T-cell responses. Serum and liver samples were collected and analyzed for HBV-specific immune responses, liver damage, and viral parameters. RESULTS:In both models of HBV infection, mice that express hepatocyte-specific small hairpin RNAs or that were given subcutaneous injections of siRNAs had reduced levels of HBV antigens, HBV replication, and viremia (1-3 log10 reduction) compared to mice given control RNAs. Vaccination induced production of HBV-neutralizing antibodies and increased numbers and functionality of HBV-specific, CD8+ T cells in mice with low, but not in mice with high, levels of HBV antigen. Mice with initially high titers of HBV and knockdown of HBV antigen expression, but not mice with reduced viremia after administration of entecavir, developed polyfunctional, HBV-specific CD8+ T cells, and HBV was eliminated. CONCLUSIONS:In mice with high levels of HBV replication, knockdown of HBV antigen expression along with a therapeutic vaccination strategy, but not knockdown alone, increased numbers of effector T cells and eliminated the virus. These findings indicate that high titers of virus antigens reduce the efficacy of therapeutic vaccination. Anti-HBV siRNAs and therapeutic vaccines are each being tested in clinical trials-their combination might cure chronic HBV infection.

Adoptive cell therapy using T cells expressing transgenic (tg) tumor antigen-targeting T cell receptors (TCRs) has become an attractive modality to treat hematological and solid cancers due to a broader array of accessible targets relative to CAR-T cell therapies. However, high-avidity TCRs specific for shared oncogenic antigens are difficult to identify. In addition, manufacturing of TCR-redirected T cells with single TCR specificity is desired to avoid mispairings and competition with endogenous chains, which can negatively impact T cell specificity and TCR expression levels. This can be achieved with CRISPR/Cas9-mediated replacement of the endogenous TCR α and β chains, by knocking out the TRAC and TRBC genes and inserting the tgTCR into the TRAC locus. While CRISPR/Cas9 genome editing has been demonstrated to be highly efficient, simultaneous edits in different loci could result in increased translocations, potentially impairing the quality and safety of the cell product. Moreover, existing cell engineering technology negatively impacts T cell quality and yield. Here, we focused on engineering T cells with specificity for Wilms' Tumor 1 (WT1), a transcription factor overexpressed by a wide range of hematological and solid tumors, that has both, restricted expression on healthy tissues and a strong correlation with oncogenesis. By applying rapid isolation technologies of WT1-specific T cells from healthy donors, we identified a lead TCR to the WT137-45 epitope, restricted to the common human leukocyte antigen, HLA-A*02:01. T cells expressing this tgTCR showed nM avidity and killed leukemia cell lines and primary acute myeloid leukemia (AML) blasts at low effector-to-target cell ratios. Epitope specificity evaluation by alanine scanning suggested that the minimal peptide recognition sequence for this TCR is restricted to WT1. Further, the lead TCR was able to activate CD8+ and CD4+ T cells, which may be beneficial for T cell persistence. By developing an improved T cell engineering process, we have achieved multiple sequential gene edits in primary human T cells, leading to knockout of the endogenous TCR with up to 99% efficiency and insertion of tgTCRs into 55-80% of the cells. This cell engineering process is scalable, adaptable to a closed system, and results in marked improvements in T cell expansion, yield, stem cell memory phenotype and T cell polyfunctionality, such as cytotoxicity, cytokine release and proliferation in response to WT1+ target cells. Additionally, the high viability profile of the process readily allows for sequential CRISPR/Cas9 gene knockout in T cells, leading to near-complete endogenous TCR removal while limiting TRAC/TRBC translocation to levels close to those found in untreated cells. T cells engineered to express the lead TCR using this process resulted in potent anti-tumor activity in vivo. Disseminated primary AML patient derived xenograft and acute lymphoblastic leukemia (ALL) cell line models were established by intravenous injection of the tumor cells in NSG or NOG mice. Animals were treated subsequently with WT1-specific or control T cells. Almost complete tumor growth inhibition in the blood and bone marrow was noted in the primary AML model. In the fast growing ALL model, WT1-T cells significantly reduced tumor burden and increased survival compared to control groups, which could be further boosted in human IL-15-expressing NOG mice vs. standard NOG mice. No signs of graph-versus-host disease (GvHD) were observed during the course of the study, which is consistent with removal of the endogenous TCR. NTLA-5001 is being advanced into clinical development for AML immunotherapy. Given the expression of WT1 in many solid tumors, engineered WT1 TCR-T cells are being further explored in those indications. Disclosures Liu: Intellia Therapeutics: Current Employment. Prodeus:Intellia Therapeutics: Current Employment. Becker:Intellia Therapeutics: Current Employment. Foisey:Intellia Therapeutics: Current Employment. Balwani:Intellia Therapeutics: Current Employment. Dutta:Intellia Therapeutics: Current Employment. Zhang:Intellia Therapeutics: Current Employment. Arredouani:Intellia Therapeutics: Current Employment. McKee:Intellia Therapeutics: Current Employment. Ciceri:Intellia Therapeutics: Membership on an entity's Board of Directors or advisory committees. Sepp-Lorenzino:Intellia Therapeutics: Current Employment. Bonini:Kiadis: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; Molmed: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; Intellia Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Schultes:Intellia Therapeutics: Current Employment, Current equity holder in publicly-traded company.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2-deoxy-2-fluoro (2-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2-F-monomer metabolites was low and transient in rats and humans. In vitro, 2-F-nucleoside 5-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for approximate to 2 years. Taken together, the results support the conclusion that 2-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

Clinical application of siRNA-based therapeutics outside of the liver has been hindered by the inefficient delivery of siRNA effector molecules into extra-hepatic organs and cells of interest. To understand the parameters that enable RNAi activity in vivo, it is necessary to develop a systematic approach to identify which cells within a tissue are permissive to oligonucleotide internalization and activity. In the present study, we evaluate the distribution and activity within the lung of chemically stabilized siRNA to characterize cell-type tropism and structure-activity relationship. We demonstrate intratracheal delivery of fully modified siRNA for RNAi-mediated target knockdown in lung CD11c(+) cells (dendritic cells, alveolar macrophages) and alveolar epithelial cells. Finally, we use an allergen-induced model of lung inflammation to demonstrate the capacity of inhaled siRNA to induce target knockdown in dendritic cells and ameliorate lung pathology.

The liver plays a central role in metabolism; however, xenobiotic metabolism variations between human hepatocytes and those in model organisms create challenges in establishing functional test beds to detect the potential drug toxicity and efficacy of candidate small molecules. In the emerging areas of RNA interference, viral gene therapy, and genome editing, more robust, long-lasting, and predictive human liver models may accelerate progress. Here, we apply a new modality to a previously established, functionally stable, multi-well bioengineered microliver-fabricated from primary human hepatocytes and supportive stromal cells-in order to advance both small molecule and nucleic acid therapeutic pipelines. Specifically, we achieve robust and durable gene silencing in vitro to tune the human metabolism of small molecules, and demonstrate its capacity to query the potential efficacy and/or toxicity of candidate therapeutics. Additionally, we apply this engineered platform to test siRNAs designed to target hepatocytes and impact human liver genetic and infectious diseases.