Besides its broad application in research and biotechnology, genome editing (GE) has great potential for clinical gene therapy, but delivery of GE tools remains a bottleneck. Whereas significant ...progress has been made in ex vivo GE delivery (e.g., by electroporation), establishment of efficient and safe in vivo delivery systems is still a challenge. Above and beyond standard vector requirements (safety, minimal/absent toxicity and immunogenicity, sufficient packaging capacity, targeting, straight and low-cost large-scale good manufacturing practice (GMP) production), GE delivery systems ideally use a hit-and-run principle to minimize off-targets as well as display of immunogenic peptides. Since currently used viral vectors do not fulfil all of these requirements, the broad variety of non-viral delivery platforms represents a promising alternative. This review provides a comprehensive analysis of the most relevant aspects of non-viral physical and chemical delivery methods in non-clinical studies and clinical trials, ranging from classic electroporation to advanced drug carriers that can transport GE tools in form of plasmid DNAs (pDNAs), mRNAs, and ribonucleoproteins (RNPs). For comparison, advantages and shortcomings of viral delivery systems are shortly discussed. In summary, we review various delivery approaches and discuss the future perspectives to use drug carriers for in vivo GE in clinical trials.
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The design of cargo carriers with high biocompatibility, unique morphological characteristics, and capability of strong bonding of fluorescent dye is highly important for the development of a ...platform for smart imaging and diagnostics. In this paper, BODIPY-doped silica nanoparticles were prepared through a "one-pot" soft-template method using a sol-gel process. Several sol-gel precursors have been used in sol-gel synthesis in the presence of soft-template to obtain the silica-based materials with the most appropriate morphological features for the immobilization of BODIPY molecules. Obtained silica particles have been shown to be non-cytotoxic and can be effectively internalized into the cervical cancer cell line (HeLa). The described method of synthesis allows us to obtain silica-based carriers with an immobilized fluorescent dye that provide the possibility for real-time imaging and detection of these carriers.
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•Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) scaffolds with SiHA were investigated.•Piezoelectric scaffolds with SiHA stimulated adhesion and differentiation of hMSCs.•Piezoelectric ...scaffolds revealed superior osteoinductive properties.•Piezoelectric scaffolds demonstrated significantly better calcium deposition potential.
In this study, bone scaffolds composed of polycaprolactone (PCL), piezoelectric poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and a combination of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and silicate containing hydroxyapatite (PHBV-SiHA) were successfully fabricated by a conventional electrospinning process. The morphological, chemical, wetting and biological properties of the scaffolds were examined. All fabricated scaffolds are composed of randomly oriented fibres with diameters from 800nm to 12μm. Fibre size increased with the addition of SiHA to PHBV scaffolds. Moreover, fibre surface roughness in the case of hybrid scaffolds was also increased. XRD, FTIR and Raman spectroscopy were used to analyse the chemical composition of the scaffolds, and contact angle measurements were performed to reveal the wetting behaviour of the synthesized materials. To determine the influence of the piezoelectric nature of PHBV in combination with SiHA nanoparticles on cell attachment and proliferation, PCL (non-piezoelectric), pure PHBV, and PHBV-SiHA scaffolds were seeded with human mesenchymal stem cells (hMSCs). In vitro study on hMSC adhesion, viability, spreading and osteogenic differentiation showed that the PHBV-SiHA scaffolds had the largest adhesion and differentiation abilities compared with other scaffolds. Moreover, the piezoelectric PHBV scaffolds have demonstrated better calcium deposition potential compared with non-piezoelectric PCL. The results of the study revealed pronounced advantages of hybrid PHBV-SiHA scaffolds to be used in bone tissue engineering.
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.
CRISPR–Cas9 is a revolutionary genome-editing technology that has enormous potential for the treatment of genetic diseases. However, the lack of efficient and safe, non-viral delivery systems has ...hindered its clinical application. Here, we report on the application of polymeric and hybrid microcarriers, made of degradable polymers such as polypeptides and polysaccharides and modified by silica shell, for delivery of all CRISPR–Cas9 components. We found that these microcarriers mediate more efficient transfection than a commercially available liposome-based transfection reagent (>70% vs. <50% for mRNA, >40% vs. 20% for plasmid DNA). For proof-of-concept, we delivered CRISPR–Cas9 components using our capsules to dTomato-expressing HEK293T cells—a model, in which loss of red fluorescence indicates successful gene editing. Notably, transfection of indicator cells translated in high-level dTomato knockout in approx. 70% of transfected cells. In conclusion, we have provided proof-of-principle that our micro-sized containers represent promising non-viral platforms for efficient and safe gene editing.
CRISPR–Cas9 is a revolutionary genome-editing technology with huge potential for research, but also clinical applications. Microcapsules combine high loading capacity with easy functionalization, e.g., to allow tissue targeting. Using an indicator cell line we show here that polymeric and hybrid silica-coated capsules mediate efficient, non-toxic and transient delivery of CRISPR–Cas9 system components. Our data suggest that microcapsules represent promising vectors for application of genome-editing tools. Display omitted
Objectives
Therapy of patients with relapsed and refractory classic Hodgkin lymphoma (r/r cHL) after PD-1 inhibitors failure remains an unresolved issue. The aim of this study was to evaluate the ...efficacy and safety of the combination of nivolumab with brentuximab vedotin (Nivo + BV) after nivolumab monotherapy failure.
Methods
This study retrospectively analyzed 21 patients with r/r cHL who were treated with the combination of Nivo + BV after Nivo failure. The response was evaluated by PET–CT scan according to the LYRIC criteria. Adverse events (AEs) were assessed according to NCI CTCAE v.4.03.
Results
Median follow-up was 19 (9–47) months. The ORR was 57%. The median OS was not reached, 24 month OS was 80% (95% CI 50–93%). Median PFS was 12 months with 24 month PFS of 31% (95% CI 12–53%). Any grade AEs were observed in 12 patients (63%), 3–4 grade AEs in 2 patients (10%). Allogeneic hematopoietic stem cell transplantation (allo-HSCT) after Nivo + BV was performed in 8 (38%) patients. The median time between Nivo + BV and allo-HSCT was 8 (5–21) months.
Conclusions
Combination of Nivo + BV in r/r cHL after nivolumab monotherapy failure is potentially an effective and safe approach.
The implementation of RNAi technology into the clinical practice has been significantly postponing due to the issues regarding to the delivery of naked siRNA predominantly to target cells. Here we ...report the approach to enhance the efficiency of siRNA delivery by encapsulating the siRNA into new carrier systems which are obtained via the combination of widely used layer-by-layer technique and in situ modification by sol-gel chemistry. We used three types of siRNAs (NP-717, NP-1155 and NP-1496) in encapsulated form as new therapeutic agents against H1N1 influenza virus infection. By employing the hybrid microcontainers for the siRNA encapsulation we demonstrate the reduction of viral nucleoprotein (NP) level and inhibition of influenza virus production in infected cell lines (MDCK and A549). The obtained hybrid carriers based on assembled biodegradable polyelectrolytes and sol-gel coating possess several advantages such as a high cell uptake efficiency, low toxicity, efficient intracellular delivery of siRNAs and the protection of siRNAs from premature degradation before reaching the target cells. These findings underpin a great potential of versatile microencapsulation technology for the development of anti-viral RNAi delivery systems against influenza virus infection.
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.
An important area in modern malignant tumor therapy is the optimization of antitumor drugs pharmacokinetics. The use of some antitumor drugs is limited in clinical practice due to their high ...toxicity. Therefore, the strategy for optimizing the drug pharmacokinetics focuses on the generation of high local concentrations of these drugs in the tumor area with minimal systemic and tissue-specific toxicity. This can be achieved by encapsulation of highly toxic antitumor drug (vincristine (VCR) that is 20–50 times more toxic than widely used the antitumor drug doxorubicin) into nano- and microcarriers with their further association into therapeutically relevant cells that possess the ability to migrate to sites of tumor. Here, we fundamentally examine the effect of drug carrier size on the behavior of human mesenchymal stem cells (hMSCs), including internalization efficiency, cytotoxicity, cell movement, to optimize the conditions for the development of carrier-hMSCs drug delivery platform. Using the malignant tumors derived from patients, we evaluated the capability of hMSCs associated with VCR-loaded carriers to target tumors using a three-dimensional spheroid model in collagen gel. Compared to free VCR, the developed hMSC-based drug delivery platform showed enhanced antitumor activity regarding those tumors that express CXCL12 (stromal cell-derived factor-1 (SDF-1)) gene, inducing directed migration of hMSCs via CXCL12 (SDF-1)/CXCR4 pathway. These results show that the combination of encapsulated antitumor drugs and hMSCs, which possess the properties of active migration into tumors, is therapeutically beneficial and demonstrated high efficiency and low systematic toxicity, revealing novel strategies for chemotherapy in the future.
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