bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2022–01–23
seventeen papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University



  1. Pharmaceutics. 2022 Jan 11. pii: 165. [Epub ahead of print]14(1):
      Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Lipid-mediated nucleic acid delivery is an alternative to viral vector-mediated gene delivery and has the following advantages. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, since transit across the nuclear membrane is not necessary, and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Delivery of RNA to target organ(s) has previously been challenging due to RNA's rapid degradation in biological systems, but cationic lipids complexed with RNA, as well as lipid nanoparticles (LNPs), have allowed for delivery and expression of the complexed RNA both in vitro and in vivo. This review will focus on the non-viral lipid-mediated delivery of RNAs, including mRNA, siRNA, shRNA, and microRNA, to the central nervous system (CNS), an organ with at least two unique challenges. The CNS contains a large number of slowly dividing or non-dividing cell types and is protected by the blood brain barrier (BBB). In non-dividing cells, RNA-lipid complexes demonstrated increased transfection efficiency relative to DNA transfection. The efficiency, timing of the onset, and duration of expression after transfection may determine which nucleic acid is best for which proposed therapy. Expression can be seen as soon as 1 h after RNA delivery, but duration of expression has been limited to 5-7 h. In contrast, transfection with a DNA lipoplex demonstrates protein expression within 5 h and lasts as long as several weeks after transfection.
    Keywords:  CHO; DNA; NIH3T3; RNA; gene delivery; lipid-mediated; molecular therapy; neurons; non-viral; post-mitotic; siRNA; transfection; transient
    DOI:  https://doi.org/10.3390/pharmaceutics14010165
  2. J Biomater Sci Polym Ed. 2022 Jan 17. 1-14
      Improving the transfection efficiency of non-viral carriers by using cationic polymers is a useful approach to addressing several challenges in gene therapy, such as cellular uptake, endosomal escape, and toxicity. Among the various cationic polymers, polydopamine (PAMAM) dendrimers have been widely utilized because of the abundance of terminal functional groups, thereby enabling further functionalization and enhancing DNA condensation and internalization into cells. The combination of various functional groups is required for these PAMAM dendrimer derivatives to function appropriately for gene delivery. Herein, we synthesized PAMAM G2-HRChol by conjugating dipeptide (histidine-arginine) and cholesterol at different ratios (6% or 23%) on the surface of PAMAM dendrimer generation 2 (PAMAM G2). Both PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% have buffering capacity, leading to improved endosomal escape after entering the cells. PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% dendrimers were condensed with pDNA to form nano-polyplexes at a weight ratio of 4 (polymer/pDNA). Polyplexes are positively charged, which facilitates cellular uptake. The transfection efficiency of PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% dendrimers was similar to that of PEI 25 kDa under optimum conditions, and the cytotoxicity was much lower than that of PEI 25 kDa in HeLa cells. In addition, after apoptin gene transfection was performed, cell death ratios of 34.47% and 22.47% were observed for PAMAM G2-HRChol 6% and PAMAM G2HRChol 23%, respectively. The results show that a suitable amount of cholesterol can improve gene transfection efficiency, and the PAMAM G2-HRChol 6% dendrimer could be a potential gene carrier in HeLa cells.
    Keywords:  PAMAM dendrimer; cholesterol; dipeptide; gene delivery; transfection
    DOI:  https://doi.org/10.1080/09205063.2022.2030657
  3. ChemMedChem. 2022 Jan 20.
      Nanoparticles consisting of a condensed nucleic acid core surrounded by protective layers which aid to overcome extracellular and intracellular hurdles to gene delivery (i.e., core-shell nanoparticles, CSNPs) synthetically mimic viruses. The outer shells shield the core and are particularly designed to enable facilitated release of the gene payload into the cytoplasm, the major limiting step in intracellular gene delivery. The hypothetical proton sponge effect and degradability in response to a stimulus (i.e., mildly acidic pH in the endosome) are two prevailing, although contested, principles in designing effective carriers for intracellular gene delivery via endosomal escape. Utilizing the highly flexible chemical-tuning of the polymeric shell via surface-initiated photo-polymerization of the various monomers at different molecular ratios, the effects of proton buffering capacity, acid-degradability, and endosomal membrane-lysis property on intracellular delivery of plasmid DNA by CSNPs were investigated. This study demonstrated the equivalently critical roles of proton buffering and acid-degradability in achieving efficient intracellular gene delivery, independent of cellular uptake. Extended proton buffering resulted in further improved transfection as long as the core structure was not compromised. The results of the study present a promising synthetic strategy to the development of an efficient, chemically-tunable gene delivery carrier.
    Keywords:  Endosomal escape, core-shell nanoparticles, nonviral vector, gene delivery
    DOI:  https://doi.org/10.1002/cmdc.202100718
  4. Pharmaceutics. 2022 Jan 17. pii: 213. [Epub ahead of print]14(1):
      The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse genetic disorders. However, for gene therapy applications, an efficient delivery vehicle is required, capable of delivering the CRISPR-Cas9 components into the cytosol of the intended target cell population. In this study, we optimized the formulation conditions of lipid nanoparticles (LNP) for delivery of ready-made CRISPR-Cas9 ribonucleic protein (RNP). The buffer composition during complexation and relative DOTAP concentrations were varied for LNP encapsulating in-house produced Cas9 RNP alone or Cas9 RNP with additional template DNA for gene correction. The LNP were characterized for size, surface charge, and plasma interaction through asymmetric flow field flow fractionation (AF4). Particles were functionally screened on fluorescent reporter cell lines for gene knock-out and gene correction. This revealed incompatibility of RNP with citrate buffer and PBS. We demonstrated that LNP for gene knock-out did not necessarily require DOTAP, while LNP for gene correction were only active with a low concentration of DOTAP. The AF4 studies additionally revealed that LNP interact with plasma, however, remain stable, whereby HDR template seems to favor stability of LNP. Under optimal formulation conditions, we achieved gene knock-out and gene correction efficiencies as high as 80% and 20%, respectively, at nanomolar concentrations of the CRISPR-Cas9 RNP.
    Keywords:  AF4; CRISPR; HDR; LNP; NHEJ; delivery; formulation
    DOI:  https://doi.org/10.3390/pharmaceutics14010213
  5. Biomolecules. 2022 Jan 08. pii: 102. [Epub ahead of print]12(1):
      Lignin is a natural renewable biomass resource with great potential for applications, while its development into high value-added molecules or materials is rare. The development of biomass lignin as potential nonviral gene delivery carriers was initiated by our group through the "grafting-from" approach. Firstly, the lignin was modified into macroinitiator using 2-bromoisobutyryl bromide. Then cationic polymer chains of poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) were grown from the lignin backbone using atom transfer radical polymerization (ATRP) to yield lignin-PDMAEMA graft copolymers (LPs) with branched structure. To gain a deep understanding of the relationship between the nonviral gene transfection efficiency of such copolymers and their structural and compositional factors, herein eight lignin-based macroinitiators with different modification degrees (MDs, from 3.0 to 100%) were synthesized. Initiated by them, a series of 20 LPs were synthesized with varied structural factors such as grafting degree (GD, which is equal to MD, determining the cationic chain number per lignin macromolecule), cationic chain length (represented by number of repeating DMAEMA units per grafted arm or degree of polymerization, DP) as well as the content of N element (N%) which is due to the grafted PDMAEMA chains and proportional to molecular weight of the LPs. The in vitro gene transfection capability of these graft copolymers was evaluated by luciferase assay in HeLa, COS7 and MDA-MB-231cell lines. Generally, the copolymers LP-12 (N% = 7.28, MD = 36.7%, DP = 13.6) and LP-14 (N% = 6.05, MD = 44.4%, DP = 5.5) showed good gene transfection capabilities in the cell lines tested. Overall, the performance of LP-12 was the best among all the LPs in the three cell lines at the N/P ratios from 10 to 30, which was usually several times higher than PEI standard. However, in MDA-MB-231 at N/P ratio of 30, LP-14 showed the best gene transfection performance among all the LPs. Its gene transfection efficiency was ca. 11 times higher than PEI standard at this N/P ratio. This work demonstrated that, although the content of N element (N%) which is due to the grafted PDMAEMA chains primarily determines the gene transfection efficiency of the LPs, it is not the only factor in explaining the performance of such copolymers with the branched structure. Structural factors of these copolymers such as grafting degree and cationic chain length could have a profound effect on the copolymer performance on gene transfection efficiency. Through carefully adjusting these factors, the gene transfection efficiency of the LPs could be modulated and optimized for different cell lines, which could make this new type of biomass-based biomaterial an attractive choice for various gene delivery applications.
    Keywords:  PDMAEMA; atom transfer radical polymerization; graft copolymer; lignin; nonviral gene carriers; plasmid DNA
    DOI:  https://doi.org/10.3390/biom12010102
  6. Pharmaceuticals (Basel). 2021 Dec 24. pii: 17. [Epub ahead of print]15(1):
      Gene therapy is a suitable alternative to chemotherapy due to the complications of drug resistance and toxicity of drugs, and is also known to reduce the occurrence of cellular mutation through the use of gene carriers. In this study, gene carrier nanoparticles with minimal toxicity and high transfection efficiency were fabricated from a biocompatible and biodegradable polymer, l-tyrosine polyurethane (LTU), which was polymerized from presynthesized desaminotyrosyl tyrosine hexyl ester (DTH) and polyethylene glycol (PEG), by using double emulsion and solvent evaporation techniques, resulting in the formation of porous nanoparticles, and then used to evaluate their potential biological activities through molecular controlled release and transfection studies. To assess cellular uptake and transfection efficiency, two model drugs, fluorescently labeled bovine serum albumin (FITC-BSA) and plasmid DNA-linear polyethylenimine (LPEI) complex, were successfully encapsulated in nanoparticles, and their transfection properties and cytotoxicities were evaluated in LX2 as a normal cell and in HepG2 and MCF7 as cancer cells. The morphology and average diameter of the LTU nanoparticles were confirmed using light microscopy, transmission electron microscopy, and dynamic light scattering, while confocal microscopy was used to validate the cellular uptake of FITC-BSA-encapsulated LTU nanoparticles. Moreover, the successful cellular uptake of LTU nanoparticles encapsulated with pDNA-LPEI and the high transfection efficiency, confirmed by gel electrophoresis and X-gal assay transfection, indicated that LTU nanoparticles had excellent cell adsorption ability, facilitated gene encapsulation, and showed the sustained release tendency of genes through transfection experiments, with an optimal concentration ratio of pDNA and LPEI of 1:10. All the above characteristics are ideal for gene carriers designed to transport and release drugs into the cytoplasm, thus facilitating effective gene therapy.
    Keywords:  DNA-LPEI complex; LTU; biodegradable; double emulsion; gene carrier; transfection
    DOI:  https://doi.org/10.3390/ph15010017
  7. Adv Sci (Weinh). 2022 Jan 17. e2104987
      Non-viral gene delivery agents, such as cationic lipids, polymers, and peptides, mainly rely on charge-based and hydrophobic interactions for the condensation of DNA molecules into nanoparticles. The human protein mitochondrial transcription factor A (TFAM), on the other hand, has evolved to form nanoparticles with DNA through highly specific protein-protein and protein-DNA interactions. Here, the properties of TFAM are repurposed to create a DNA transfection agent by means of protein engineering. TFAM is covalently fused to Listeria monocytogenes phospholipase C (PLC), an enzyme that lyses lipid membranes under acidic conditions, to enable endosomal escape and human vaccinia-related kinase 1 (VRK1), which is intended to protect the DNA from cytoplasmic defense mechanisms. The TFAM/DNA complexes (TFAMoplexes) are stabilized by cysteine point mutations introduced rationally in the TFAM homodimerization site, resulting in particles, which show maximal activity when formed in 80% serum and transfect HeLa cells in vitro after 30 min of incubation under challenging cell culture conditions. The herein developed TFAM-based DNA scaffolds combine interesting characteristics in an easy-to-use system and can be readily expanded with further protein factors. This makes the TFAMoplex a promising tool in protein-based gene delivery.
    Keywords:  DNA nanoparticles; non-viral gene delivery; protein engineering; protein-based DNA carrier
    DOI:  https://doi.org/10.1002/advs.202104987
  8. Eur J Pharm Sci. 2022 Jan 13. pii: S0928-0987(22)00010-0. [Epub ahead of print] 106125
      Vascular endothelial growth factor (VEGF) is considered as one of the vital growth factors for angiogenesis, which is primarily responsible for the progress and maintenance of new vascular network in tumor. Numerous studies report that inhibition of VEGF-induced angiogenesis is a potent technique for cancer suppression. Recently, RNA interference, especially small interfering RNA (siRNA) signified a promising approach to suppress the gene expression. However, the clinical implementation of biological macromolecules such as siRNA is significantly limited because of stability and bioavailability issues. Herein, self-assembled peptide nanospheres have been generated from L,L-cyclic peptides using hydrophobic (Trp), positively charged (Arg) and cysteine (Cys) amino acid residues and demonstrated as vehicles for intracellular delivery of VEGF siRNA and VEGF antisense oligonucleotide. Formation of peptide nanostructures is confirmed by HR-TEM, AFM, SEM and DLS analysis. Possible mechanism of self-assembly of the cyclic peptides and their binding with macromolecules are demonstrated by in-silico analysis. Gel electrophoresis reveals that the newly generated peptide based organic materials exhibit strong binding affinity toward siRNAs / antisense oligonucleotides (ASOs) at optimum concentration. Flow cytometry and confocal microscopy results confirm the efficiency of the new biomaterials toward the intracellular delivery of fluorescent labeled siRNA / ASOs. Furthermore, VEGF expression evaluated by western blot and RT-PCR upon the delivery of functional VEGF siRNA/ASOs suggests that very low concentrations of VEGF siRNA/ASOs cause significant gene knockdown at protein and mRNA levels, respectively.
    Keywords:  Antisense oligonucleotides; Cyclic peptide; Gene knockdown; In-silico; Nanostructures; VEGF; siRNA
    DOI:  https://doi.org/10.1016/j.ejps.2022.106125
  9. Drug Deliv. 2022 Dec;29(1): 316-327
      Ultrasound nanodroplets (NDs) have been reported as a promising nanocarrier for siRNA delivery depending on its unique strengths of sonoporation. Presently, common means for NDs-mediated siRNA delivery is through electrostatic interaction, but challenges like cationic toxicity still exist. In this study, we demonstrated a novel strategy to construct negatively charged and ultrasound (US)-responsive O-carboxymethyl chitosan (O-CMS) NDs as a siRNA targeted delivery system through three-way junction of bacteriophage phi29 DNA packaging motor (3WJ-pRNA) nanotechnology. 39nt A10-3.2 aptamer targeting prostate specific membrane antigen (PSMA) and 21nt siRNA against cationic amino acid transporter 1 (siCAT-1) were annealed to 3WJ-pRNA scaffold via complementation with an extended sequence. The cholesterol molecule attached to one branch facilitates the 3WJ-pRNA nanoparticles anchoring onto NDs. The desired O-CMS NDs with siRNA-loading and RNA-aptamer modification (A10-3.2/siCAT-1/3WJ-NDs) were successfully prepared, which were with spherical shapes, core-shell structures and uniform in sizes (198 nm with PDI 0.3). As a main proportion of shell, O-CMC showed a certain anti-tumor effects. In vitro studies demonstrated that A10-3.2/siCAT-1/3WJ-NDs exhibited good contrast-enhanced US imaging, buffering capacity and high bio-safety, were able to deliver siCAT-1 to PSMA-overexpressed prostate cancer cells under US irradiation, thus silence the CAT-1 expression, and consequently suppressing 22RV1 cell proliferation and migration. Taken overall, our findings provide a promising strategy to develop negatively charged and US-responsive NDs for tumor-targeted siRNA delivery.
    Keywords:  Ultrasound nanodroplet; gene nanotechnology; negatively charged; siRNA delivery; tumor-targeting
    DOI:  https://doi.org/10.1080/10717544.2022.2026532
  10. Pharmaceutics. 2022 Jan 11. pii: 162. [Epub ahead of print]14(1):
      Rheumatoid arthritis (RA) is one of the most common autoimmune diseases worldwide, causing severe cartilage damage and disability. Despite the recent progress made in RA treatment, limitations remain in achieving early and efficient therapeutic intervention. Advanced therapeutic strategies are in high demand, and siRNA-based therapeutic technology with a gene-silencing ability represents a new approach for RA treatment. In this study, we created a cationic delivery micelle consisting of low-molecular-weight (LMW) polyethylenimine (PEI)-cholesterol-polyethylene glycol (PEG) (LPCE) for small interfering RNA (siRNA)-based RA gene therapy. The carrier is based on LMW PEI and modified with cholesterol and PEG. With these two modifications, the LPCE micelle becomes multifunctional, and it efficiently delivered siRNA to macrophages with a high efficiency greater than 70%. The synthesized LPCE exhibits strong siRNA protection ability and high safety. By delivering nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 siRNA, the p65 siRNA/LPCE complex efficiently inhibited macrophage-based cytokine release in vitro. Local administration of the p65 siRNA/LPCE complex exhibited a fast and potent anti-inflammatory effect against RA in a mouse model. According to the results of this study, the functionalized LPCE micelle that we prepared has potential gene therapeutic implications for RA.
    Keywords:  gene therapy; micelle; p65; rheumatoid arthritis; siRNA
    DOI:  https://doi.org/10.3390/pharmaceutics14010162
  11. Chem Commun (Camb). 2022 Jan 18.
      Topological structure plays a critical role in gene delivery of cationic polymers. Cyclic poly(β-amino ester)s (CPAEs) are successfully synthesized via sequential Michael addition and free radical initiating ring-closure reaction. The CPAEs exhibit superior gene transfection efficiency and safety profile compared to their linear counterparts.
    DOI:  https://doi.org/10.1039/d1cc06480k
  12. ACS Appl Mater Interfaces. 2022 Jan 18.
      Microglia are the major innate immune cells in the brain and are essential for maintaining homeostasis in a neuronal microenvironment. Currently, a genetic tool to modify microglial gene expression in specific brain regions is not available. In this report, we introduce a tailor-designed method that uses lipid and polymer hybridized nanoparticles (LPNPs) for the local delivery of small interfering RNAs (siRNAs), allowing the silencing of specific microglial genes in the hypothalamus. Our physical characterization proved that this LPNP-siRNA was uniform and stable. We demonstrated that, due to their natural phagocytic behavior, microglial cells are the dominant cell type taking up these LPNPs in the hypothalamus of rats. We then tested the silencing efficiency of LPNPs carrying a cluster of differentiation molecule 11b (CD11b) or Toll-like receptor 4 (TLR4) siRNA using different in vivo and in vitro approaches. In cultured microglial cells treated with LPNP-CD11b siRNA or LPNP-TLR4 siRNA, we found a silencing efficiency at protein expression levels of 65 or 77%, respectively. In line with this finding, immunohistochemistry and western blotting results from in vivo experiments showed that LPNP-CD11b siRNA significantly inhibited microglial CD11b protein expression in the hypothalamus. Furthermore, following lipopolysaccharide (LPS) stimulation of cultured microglial cells, gene expression of the TLR4 downstream signaling component myeloid differentiation factor 88 and its associated cytokines was significantly inhibited in LPNP-TLR4 siRNA-treated microglial cells compared with cells treated with LPNP-scrambled siRNA. Finally, after LPNP-TLR4 siRNA injection into the rat hypothalamus, we observed a significant reduction in microglial activation in response to LPS compared with the control rats injected with LPNP-scrambled siRNA. Our results indicate that LPNP-siRNA is a promising tool to manipulate microglial activity locally in the brain and may serve as a prophylactic approach to prevent microglial dysfunction-associated diseases.
    Keywords:  CD11b; TLR4; hypothalamus; microglia; nanoparticles; phagocytosis; siRNA
    DOI:  https://doi.org/10.1021/acsami.1c22434
  13. ACS Appl Mater Interfaces. 2022 Jan 21.
      Pulmonary delivery of anti-inflammatory siRNA presents a promising approach for localized therapy of acute lung injury (ALI), while polycationic vectors can be easily trapped by the negatively charged airway mucin glycoproteins and arbitrarily internalized by epithelial cells with nontargetability for immunological clearance. Herein, we report a material, the dopamine (DA)-grafted hyaluronic acid (HA-DA), coating on an anti-TNF-α vector to address these limitations. HA-DA was simply synthesized and facilely coated on poly(β-amino ester) (BP)-based siRNA vectors via electrostatic attraction. The resulting HA-DA/BP/siRNA displayed significantly enhanced mucus penetration, attributable to the charge screen effect of HA-DA and the bioadhesive nature of the grafting DA. After transmucosal delivery, the nanosystem could target diseased macrophages via CD44-mediated internalization and rapidly escape from endo/lysosomes through the proton sponge effect, resulting in effective TNF-α regulation. Meanwhile, DA modification endowed the coating material with robust antioxidative capability to scavenge a broad spectrum of reactive oxygen/nitrogen species (RONS), which protected the lung tissue from oxidative damage and synergized with anti-TNF-α to inhibit a cytokine storm. As a result, a remarkable amelioration of ALI was achieved in a lipopolysaccharide (LPS)-stimulated mice model. This study provides a multifunctional coating material to facilitate pulmonary drug delivery for the treatment of lung diseases.
    Keywords:  core−shell nanoparticles; cytokines; gene vector; macrophages; targeting delivery
    DOI:  https://doi.org/10.1021/acsami.1c23069
  14. J Control Release. 2022 Jan 15. pii: S0168-3659(22)00030-X. [Epub ahead of print]342 362-371
      Harnessing RNA-based therapeutics for cancer, inflammation, and viral diseases is hindered by poor delivery of therapeutic RNA molecules. Targeting leukocytes to treat these conditions holds great promise, as they are key participants in their initiation, drug response, and treatment. The various extra- and intra-cellular obstacles that impediment the clinical implementation of therapeutic RNA can be overcome by utilizing drug delivery systems. However, delivery of therapeutic RNA to leukocytes poses an even greater challenge as these cells are difficult to reach and transfect upon systemic administration. This review briefly describes the existing successful delivery strategies that efficiently target leukocytes in vivo and discuss their potential clinical applicability.
    Keywords:  Drug delivery; Leukocytes; Nanocarriers; RNA; mRNA
    DOI:  https://doi.org/10.1016/j.jconrel.2022.01.016
  15. Front Med Technol. 2020 ;2 602236
      Non-viral gene therapy of the brain is enabled by the development of plasmid DNA brain delivery technology, which requires the engineering and manufacturing of nanomedicines that cross the blood-brain barrier (BBB). The development of such nanomedicines is a multi-faceted problem that requires progress at multiple levels. First, the type of nanocontainer, e.g., nanoparticle or liposome, which encapsulates the plasmid DNA, must be developed. Second, the type of molecular Trojan horse, e.g., peptide or receptor-specific monoclonal antibody (MAb), must be selected for incorporation on the surface of the nanomedicine, as this Trojan horse engages specific receptors expressed on the BBB, and the brain cell membrane, to trigger transport of the nanomedicine from blood into brain cells beyond the BBB. Third, the plasmid DNA must be engineered without bacterial elements, such as antibiotic resistance genes, to enable administration to humans; the plasmid DNA must also be engineered with tissue-specific gene promoters upstream of the therapeutic gene, to insure gene expression in the target organ with minimal off-target expression. Fourth, upstream manufacturing of the nanomedicine must be developed and scalable so as to meet market demand for the target disease, e.g., annual long-term treatment of 1,000 patients with an orphan disease, short term treatment of 10,000 patients with malignant glioma, or 100,000 patients with new onset Parkinson's disease. Fifth, downstream manufacturing problems, such as nanomedicine lyophilization, must be solved to ensure the nanomedicine has a commercially viable shelf-life for treatment of CNS disease in humans.
    Keywords:  blood-brain barrier; insulin receptor; liposomes; mnoclonal antibody; nanoparticles; non-viral gene therapy; transferrin receptor
    DOI:  https://doi.org/10.3389/fmedt.2020.602236
  16. Biomaterials. 2022 Jan 03. pii: S0142-9612(21)00712-2. [Epub ahead of print]281 121356
      The repeated administration of non-degradable dendrimers can lead to toxicity due to their bioaccumulation. Furthermore, in drug delivery applications, carrier stability can result in low biological performance due to insufficient intracellular cargo release. A novel family of versatile, biosafe, water-soluble, and fully biodegradable PEG-dendritic nanosystems is proposed, which overcomes the limitations of the most used dendrimers. Their novelty relies on the full and adjustable degradability thanks to the presence of tunable ester bonds in every dendritic arm. These dendritic nanosystems present peripheral azides that allow their easy multivalent functionalization, by "click" chemistry, with a vast range of ligands to act as versatile carriers. Here, their amine-functionalization to serve as nucleic acid vectors for gene therapy is explored. These nanosystems complex and protect efficiently siRNA in very small dendriplexes (<60 nm), being successfully cell-internalized, including in hard-to-transfect neuronal cells even when in full tissue explants (dorsal root ganglia). Importantly, full biodegradability was crucial for an efficient nucleic acid intracellular release and the attainment of excellent transfection efficiencies. The reported fully biodegradable dendritic nanosystems can act as multi-function nanotherapeutics for gene therapy, and also for broader applications in nanomedicine. Therefore, they represent top-notch and clinically translatable health facilitating nanotechnologies for further developments in theranostics.
    Keywords:  Biodegradability; Dendrimers; Nanomedicine; Neuronal cells; siRNA
    DOI:  https://doi.org/10.1016/j.biomaterials.2021.121356
  17. Sci Adv. 2022 Jan 21. 8(3): eabj6901
      Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long-lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP-mediated CRISPR-Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding-associated phenotypes were recovered in both hemophilia A and B mice. No active off-targets, liver-induced toxicity, and substantial anti-Cas9 immune responses were detected, indicating that the LNP-mediated CRISPR-Cas9 delivery was a safe and efficient approach for hemophilia therapy.
    DOI:  https://doi.org/10.1126/sciadv.abj6901