Advances in molecular biology have revolutionized the use of messenger RNA (mRNA) as a therapeutic. The concept of nucleic acid therapy with mRNA originated in 1990 when Wolff et al. reported successful expression of proteins in target organs by direct injection of either plasmid DNA or mRNA. It took decades to bring the transfection efficiency of mRNA closer to that of DNA. The next few decades were dedicated to turning in vitro-transcribed (IVT) mRNA from a promising delivery tool for gene therapy into a full-blown therapeutic modality, which changed the biotech market rapidly. Hundreds of clinical trials are currently underway using mRNA for prophylaxis and therapy of infectious diseases and cancers, in regenerative medicine, and genome editing. The potential of IVT mRNA to induce an innate immune response favors its use for vaccination and immunotherapy. Nonetheless, in non-immunotherapy applications, the intrinsic immunostimulatory activity of mRNA directly hinders the desired therapeutic effect since it can seriously impair the target protein expression. Targeting the same innate immune factors can increase the effectiveness of mRNA therapeutics for some indications and decrease it for others, and vice versa. The review aims to present the innate immunity-related ‘barriers’ or ‘springboards’ that may affect the development of immunotherapies and non-immunotherapy applications of mRNA medicines.

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Gene therapy is a fast developing field of medicine with hundreds of ongoing early-stage clinical trials and numerous preclinical studies. Genome editing (GE) now is an increasingly important technology for achieving stable therapeutic effect in gene correction, with hematopoietic cells representing a key target cell population for developing novel treatments for a number of hereditary diseases, infections and cancer. By introducing a double strand break (DSB) in the defined locus of genomic DNA, GE tools allow to knockout the desired gene or to knock-in the therapeutic gene if provided with an appropriate repair template. Currently, the efficiency of methods for GE-mediated knock-in is limited. Significant efforts were focused on improving the parameters and interaction of GE nuclease proteins. However, emerging data suggests that optimal characteristics of repair templates may play an important role in the knock-in mechanisms. While viral vectors with notable example of AAVs as a donor template carrier remain the mainstay in many preclinical trials, non-viral templates, including plasmid and linear dsDNA, long ssDNA templates, single and double-stranded ODNs, represent a promising alternative. Furthermore, tuning of editing conditions for the chosen template as well as its structure, length, sequence optimization, homology arm (HA) modifications may have paramount importance for achieving highly efficient knock-in with favorable safety profile. This review outlines the current developments in optimization of templates for the GE mediated therapeutic gene correction.

Introduction: Gene therapy is a breakthrough medical field which focuses on the therapeutic delivery of recombinant nucleic acids in order to treat or prevent a broad spectrum of diseases. However, a number of important obstacles remain before its wide introduction into clinical practice can be envisaged. One of the biggest bottlenecks is the lack of efficient and safe delivery technologies, particularly, for in vivo distribution. Above and beyond standard requirements for carriers, the delivery systems for gene therapy ideally use a hit-and-run principle (to minimize off-target effect and display of immunogenic moieties). None of the currently used viral vectors fulfills all of these requirements. Therefore, the growing variety of non-viral delivery platforms represents a promising alternative. Areas covered: This review summarizes the Layer-by-Layer (LbL) approaches that can be effectively used for the gene delivery, considering various examples with the transfer of pDNA, mRNA, siRNA as well as genome-editing tools. Ex vivo gene modification of clinically relevant cells and clinical aspects for possible application of LbL systems in gene therapy are also underlined. Expert opinion: The LbL technique provides broad opportunities for the delivery of genetic material for various purposes and offers promise for future clinical application in gene therapy.

To date, the impact of the tumor microenvironment on the prognosis of patients with classic Hodgkin lymphoma (cHL) during anti-PD-1 therapy has been studied insufficiently. This retrospective study included 61 primary samples of lymph nodes from patients who had relapsed/refractory (r/r) cHL and were treated with nivolumab. Repeated samples were obtained in 15 patients at relapse or disease progression after immunotherapy. Median follow-up was 55 (13–63) months. The best overall response rate and progression-free survival (PFS) were analyzed depending on the expression of CD68, CD163, PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CD163/c-maf in the tumor microenvironment in primary and sequential biopsies. The combination of CD163/c-maf antibodies was used for the identification of M2 macrophages (M2). A low number of macrophages in primary samples was associated with inferior PFS during nivolumab treatment (for CD163-positive cells p = 0.0086; for CD68-positive cells p = 0.037), while a low number of M2 with higher PFS (p = 0.014). Complete response was associated with a lower level of M2 (p = 0.011). In sequential samples (before and after nivolumab therapy) an increase in PD-1 (p = 0.011) and LAG-3 (p = 0.0045) and a depletion of CD68 (p = 0.057) and CD163 (p = 0.0049)-positive cells were observed. The study expands understanding of the cHL microenvironment structure and dynamics during nivolumab therapy in patients with r/r cHL.