Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides.

The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.

Appearance of IgG to SARS-CoV-2 in saliva effectively indicates seroconversion in mRNA vaccinated immunocompromised individuals

BackgroundImmunocompromised individuals are highly susceptible to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Whether vaccine-induced immunity in these individuals involves the oral cavity, a primary site of infection, is presently unknown. MethodsImmunocompromised individuals (n=404) and healthy controls (n=82) participated in a prospective clinical trial encompassing two doses of the mRNA BNT162b2 vaccine. Immunocompromised individuals included primary immunodeficiencies (PID) and secondary immunodeficiencies caused by human immunodeficiency virus (HIV) infection, allogeneic hematopoietic stem cell transplantation (HSCT)/chimeric antigen receptor T cell therapy (CAR-T), solid organ transplantation (SOT), and chronic lymphocytic leukemia (CLL). Saliva and serum samples were collected at four time points from the first vaccine dose until 2 weeks after second dose. SARS-CoV-2 spike specific immunoglobulin G (IgG) responses were quantified by a multiplex bead-based assay in saliva and correlated to paired serum IgG titers determined by Elecsys(R) Anti-SARS-CoV-2 S assay. ResultsIgG responses to the SARS-CoV-2 spike full-length trimeric glycoprotein (Spike-f) and S1 subunit in saliva in the HIV and HSCT/CAR-T groups were comparable to healthy controls. In contrast, PID, SOT, and CLL patients all displayed weaker responses which were mainly influenced by disease parameters or immunosuppressants. Salivary IgG levels strongly correlated with serum IgG titers on days 21 and 35 (rho=0.8079 and 0.7768, p=<0.0001). Receiver operating characteristic curve analysis for the predictive power of salivary IgG yielded AUC=0.95, PPV=90.7% for the entire cohort on D35. ConclusionsSaliva conveys humoral responses induced by BNT162b2 vaccination. The predictive power makes it highly suitable for screening low responding/vulnerable groups for revaccination. Trial RegistrationClinicalTrials.gov Identifier: NCT04780659 FundingKnut and Alice Wallenberg Foundation, Erling Perssons family foundation, Region Stockholm, Swedish Research Council, Karolinska Institutet, The Swedish Blood Cancer Foundation and the organization for PID patient group in Sweden, and Nordstjernan AB. Center for Medical Innovation (CIMED), the Swedish Medical Research Council and the Stockholm County Council (ALF). GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/21264377v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1428efcorg.highwire.dtl.DTLVardef@b97e88org.highwire.dtl.DTLVardef@224661org.highwire.dtl.DTLVardef@3ab25a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Safety and efficacy of the mRNA BNT162b2 vaccine against SARS-CoV-2 in five groups of immunocompromised patients and healthy controls in a prospective open-label clinical trial

BackgroundPatients with immunocompromised disorders have mainly been excluded from clinical trials of vaccination against COVID-19. Thus, the aim of this prospective clinical trial was to investigate the safety and efficacy after two doses of BNT162b2 mRNA vaccination in five selected groups of immunocompromised patients and healthy controls. Methods539 study subjects (449 patients and 90 controls) were included in the clinical trial. The patients had either primary (n=90), or secondary immunodeficiency disorders due to human immunodeficiency virus infection (n=90), allogeneic hematopoietic stem cell transplantation/chimeric antigen receptor T cell therapy (n=90), solid organ transplantation (SOT) (n=89), or chronic lymphocytic leukemia (CLL) (n=90). The primary endpoint was seroconversion rate two weeks after the second dose. The secondary endpoints were safety and documented SARS-CoV-2 infection. FindingsAdverse events were generally mild, but one case of fatal suspected unexpected serious adverse reaction occurred. 72{middle dot}2% of the immunocompromised patients seroconverted compared to 100% of the controls (p=0.004). Lowest seroconversion rates were found in the SOT (43{middle dot}4%) and CLL (63{middle dot}3%) patient groups with observed negative impact of treatment with mycophenolate mofetil and ibrutinib, respectively. InterpretationThe results showed that the mRNA BNT162b2 vaccine was safe in immunocompromised patients. The rate of seroconversion was substantially lower than in healthy controls, with a wide range of rates and antibody titres among predefined patient groups and subgroups. This clinical trial highlights the need for additional vaccine doses in certain immunocompromised patient groups and/or subgroups to improve immunity. FundingKnut and Alice Wallenberg Foundation, Nordstjernan AB, Region Stockholm, Swedish Research Council, Karolinska Institutet, and organizations for PID/CLL-patients in Sweden.

[Gene therapy now enters many clinical disciplines]. / Genterapi kommer nu inom många medicinska specialiteter.

During the last three years the development of gene therapy has been rapid. The number of gene therapies on the market has more than doubled and within certain disease areas, such as hemophilia, a large proportion of patients will likely be successfully treated. The implementation will be demanding for the Swedish health care system, owing to the very high cost of these drugs, but total costs may be reduced, especially if a single treatment is sufficient. New disruptive technologies such as gene editing are being applied in clinical trials in disease areas such as hemoglobinopathies.