bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2022‒10‒02
twelve papers selected by
the Merkel lab
Ludwig-Maximilians University

  1. J Control Release. 2022 Sep 21. pii: S0168-3659(22)00635-6. [Epub ahead of print]
      RNA interference (RNAi) is a major cellular mechanism regulating gene expression in which short double-stranded RNA molecules called small interfering RNA (siRNA) mediate sequence-specific mRNA degradation. RNAi technology has recently emerged as a promising therapeutic platform for the effective treatment of various diseases caused by inappropriate gene activity, such as cancer. However, the clinical translation of siRNA therapeutics has been hampered by the major hurdles associated with biological instability and limited delivery efficiency. Based on the various efforts, recent siRNA delivery strategies using cationic lipids and polymers allowed to enhance pharmacokinetics and delivery efficiency, resulting in potent and liver-targeted RNAi therapy. However, non-specific protein adsorption, high liver accumulation, and severe toxicity of cationic nanocarriers still limit the possibility of transfer of siRNA therapeutics from the laboratory to the clinic. One of the promising delivery strategies to overcome the limitations of siRNA therapeutics is carrier-free bioconjugation which is chemically modified and connected with biocompatible molecules such as lipids, peptides, antibodies, aptamers, and polymers. These molecularly engineered siRNA conjugates can be utilized for RNAi delivery to tissues beyond the liver, providing opportunities for clinical translation. This review focused on introducing the recent progress in molecularly engineered siRNA conjugates and their applications toward overcoming the limitations of siRNA for tumor-targeted delivery and therapy.
    Keywords:  Gene delivery; RNA interference; Small interfering RNA; cancer therapy; siRNA conjugates
  2. Nanoscale. 2022 Sep 28.
      Proinflammatory cytokines such as Tumor Necrosis Factor-α (TNF-α) are critical mediators of inflammatory bowel disease pathogenesis, and are important targets to restore intestinal homeostasis. Herein, we present the engineering and screening of gemini lipid nanoparticles (GLNPs) for siRNA delivery to colon epithelial cells, macrophages and dendritic cells, and their ability to deliver siRNA therapeutics to the inflamed gastrointestinal tract. We synthesized eight gemini cationic lipids by tethering two lithocholic acid molecules through 3'-hydroxyl- and 24'-carboxyl-derived ammonium groups using different polyalkylene spacers. Screening of GLNPs, composed of gemini cationic lipid and dioleoylphosphatidylethanolamine lipid, showed that GLNPs derived from gemini lipid G1 are the most effective in the delivery of siRNA across mammalian cell membranes with reduced toxicity. Gemini lipid G1-derived siRNA-GLNP complexes (siGLNPs) can effectively reduce gene expression, and are stable in simulated gastric fluid. The delivery of TNF-α siRNA using siGLNPs can mitigate gut inflammation in a dextran sodium sulfate-induced murine inflammation model. As CD4+ T cells, especially Th17 cells, are key mediators of gut inflammation, we further showed that these siGLNPs inhibit infiltration and differentiation of CD4+ T cells to Th17 and Treg cells. Therefore, this study highlights the potential of GLNPs derived from lithocholic acid-derived gemini cationic lipids for the development of next-generation nucleic acid delivery vehicles.
  3. J Mater Chem B. 2022 Sep 27.
      Small interfering RNA (siRNA) has increasingly evolved as a potent therapeutic solution for several pathological conditions including cancers via post-transcriptional oncogene suppression in cellular pathways. And, the key for siRNA-based therapy highly relies on the successful siRNAs delivery into the target cells, which is significantly challenged by their instability, poor cellular uptake and targeting capability. To overcome these issues, herein, a new type of RNA nanostructure, the bivalent aptamer and terminus-free siRNA junction, is synthesized and employed for effective gene silencing in cancer cells. Such a siRNA junction can be readily prepared by the self-assembly of three RNA sequences and subsequent ligation of the nicks. The as-synthesized siRNA junction shows highly improved enzymatic stability and targeting capability and can be efficiently delivered into the target cells to induce cell apoptosis. With these integrated advantages, the siRNA junction can therefore offer new potentials for the design of different siRNA therapeutics for various diseases.
  4. Mol Pharm. 2022 Sep 26.
      Within the field of lipid nanoparticles (LNPs) for RNA delivery, the focus has been mainly placed on organ level delivery, which can mask cellular level effects consequential to therapeutic applications. Here, we studied a pair of LNPs with similar physical properties and discovered how the chemistry of the ionizable amino lipid can control the endogenous LNP identity, affecting cellular uptake in the liver and altering therapeutic outcomes in a model of liver cancer. Although most LNPs accumulate in the liver after intravenous administration (suggesting that liver delivery is straightforward), we observed an unexpected behavior when comparing two similar LNP formulations (5A2-SC8 and 3A5-SC14 LNPs) that resulted in distinct RNA delivery within the organ. Despite both LNPs possessing similar physical properties, ability to silence gene expression in vitro, strong accumulation within the liver, and a shared pKa of 6.5, only 5A2-SC8 LNPs were able to functionally deliver RNA to hepatocytes. Factor VII (FVII) activity was reduced by 87%, with 5A2-SC8 LNPs carrying FVII siRNA (siFVII), while 3A5-SC14 LNPs carrying siFVII produced baseline FVII activity levels comparable to the nontreatment control at a dosage of 0.5 mg/kg. Protein corona analysis indicated that 5A2-SC8 LNPs bind apolipoprotein E (ApoE), which can drive LDL-R receptor-mediated endocytosis in hepatocytes. In contrast, the surface of 3A5-SC14 LNPs was enriched in albumin but depleted in ApoE, which likely led to Kupffer cell delivery and detargeting of hepatocytes. In an aggressive MYC-driven liver cancer model relevant to hepatocytes, 5A2-SC8 LNPs carrying let-7g miRNA were able to significantly extend survival up to 121 days. Since disease targets exist in an organ- and cell-specific manner, the clinical development of RNA LNP therapeutics will require an improved understanding of LNP cellular tropism within organs. The results from our work illustrate the importance of understanding the cellular localization of RNA delivery and incorporating further checkpoints when choosing nanoparticles beyond biochemical and physical characterization, as small changes in the chemical composition of LNPs can have an impact on both the biofate of LNPs and therapeutic outcomes.
    Keywords:  RNA delivery; cancer therapy; cell tropism; lipid nanoparticles; protein corona
  5. Acta Biomater. 2022 Sep 23. pii: S1742-7061(22)00615-8. [Epub ahead of print]
      Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system adapted from bacteria is a programmable nuclease-based genome editing tool. The long-lasting effect of gene silencing or correction is beneficial in cancer treatment. Considering the need to broaden the practical application of this technology, highly efficient non-viral vectors are urgently required. We prepared a multifunctional non-viral vector that could actively target tumor cells and deliver CRISPR/Cas9 plasmids into nuclei of cancer cells. Protamine sulfate (PS) which contains nuclear localization sequence was utilized to condense plasmid DNA and facilitate nuclei-targeted delivery. Liposome-coated protein/DNA complex avoided the degradation of nuclease in blood circulation. The obtained PS@Lip/pCas9 was further modified with distearoyl phosphoethanolamine-polyethylene glycol-hyaluronic acid (HA) to endow the vector ability to actively target tumor cell. Results suggested that PS@HA-Lip could deliver CRISPR/Cas9 plasmids into nuclei of tumor cells and induce genome editing effect. With the disruption of MTH1 (mutT homolog1) gene, the growth of non-small cell lung cancer was inhibited. Moreover, cell apoptosis in tumor tissue was promoted, and liver metastasis of non-small cell lung cancer (NSCLC) was reduced. Our study has provided a therapeutic strategy targeting MTH1 gene for NSCLC therapy. STATEMENT OF SIGNIFICANCE: CRISPR/Cas9 as a powerful tool for genome editing has drawn much attention. The long-lasting effect possesses unique advantage in cancer treatment. Non-viral vectors have high loading capacity, high safety and low immunogenicity, playing an important role in CRISPR/Cas9 delivery. In our study, a multifunctional non-viral vector for the efficient delivery of CRISPR/Cas9 plasmid was constructed. With the active targeting ligand and nuclei-targeting component, the cargo was efficiently delivered into cell nuclei and exerted genome editing effect. By using this vector, we successfully inhibited the growth and induced the apoptosis of non-small cell lung cancer by disrupting MTH1 expression with good safety. Our work provided an efficient non-vial vector for CRISPR/Cas9 delivery and explored the possibility for cancer treatment.
    Keywords:  CRISPR/Cas9; non-viral vector; targeted gene therapy; targeted nano delivery; tumor
  6. Int J Pharm. 2022 Sep 22. pii: S0378-5173(22)00777-3. [Epub ahead of print] 122223
      Lipid/polymer hybrid nanoparticles loaded with red fluorescent protein (RFP) encoded plasmid DNA (pDNA) was formulated using poly-lactic-co-glycolic acid (PLGA), cationic lipid DC-cholesterol and surfactant mPEG2000- DSPE. A lipid/ polymer ratio of 1: 10 at 1 mg/ mL surfactant concentration was found to be optimal for producing nanoparticles with diameters of 100- 120 nm that remained stable upon ultracentrifugation. The production of lipid/ polymer hybrid nanoparticles was investigated using microfluidics with a toroidal mixer design. Our results showed that the flow parameters significantly influenced the physicochemical characteristics of nanoparticles and loading of pDNA was only achieved at flow rate ratio (FRR) of 3: 1. The pDNA associated with nanoparticles was demonstrated to be structurally intact using gel electrophoresis, and the encapsulation efficiency (EE) was measured to be ∼65%. The prepared hybrid nanoparticles resulted in 20% of transfection efficacy in human embryonic kidney cells (HEK293T). This study demonstrated the potential of microfluidics in the development of hybrid nanoparticles for pDNA delivery, thus facilitating the clinical translation of DNA therapeutics.
    Keywords:  PLGA; gene therapy; microfluidics; nanoparticles; plasmid DNA
  7. J Comput Chem. 2022 Sep 28.
      Polyethyleneimine (PEI), one of the non-viral vectors of great interest for gene delivery, was investigated at all-atom level, with particular emphasis on its branched form. We report the extension of our previously published CHARMM force field (FF) for linear PEI, with parameters optimized specifically for branched configurations. A new residue type for the branch connector is introduced and the charges and bonded parameters are derived from ab initio calculations based on a model polymer. The new FF is validated by extensive molecular dynamics simulations of solvated branched PEIs of various protonation fractions and branch lengths. The gyration radii, end-to-end distances, and diffusion coefficients are compared with results for linear PEIs of similar molecular weights and protonation patterns. Solvated complexes of DNA with (linear/branched) PEI were also investigated to determine favorable attachment conformations. The parametrized atomistic force field is suitable for simulations of PEI with arbitrary branching pattern, protonation, and size, and is expected to provide relevant insights regarding optimal conditions for DNA-PEI complex formation.
    Keywords:  atomistic force fields; cationic polymers; molecular dynamics; polyethyleneimine
  8. J Control Release. 2022 Sep 26. pii: S0168-3659(22)00641-1. [Epub ahead of print]
      CRISPR/Cas9 gene-editing technology shows great potential for treating a variety of diseases, such as glioblastoma multiforme (GBM). However, CRISPR components suffer from inherent delivery challenges, such as poor in vivo stability of Cas9 protein and gRNA, low blood-brain barrier (BBB) permeability and non-specific tissue or cell targeting. These defects have limited the application of Cas9/gRNA ribonucleoprotein (RNP) complexes for GBM therapy. Here, we developed a brain-targeted CRISPR/Cas9 based nanomedicine by fabricating an angiopep-2 decorated, guanidinium and fluorine functionalized polymeric nanoparticle with loading Cas9/gRNA RNP for the treatment of GBM. The guanidinium and fluorine domains of our polymeric nanoparticles were both capable of interacting with Cas9/gRNA RNP to stabilize it in blood circulation, without impairing its activity. Moreover, by leveraging angiopep-2 peptide functionality, the RNP nanoparticles efficiently crossed the BBB and accumulated in brain tumors. In U87MG cells, we achieved approximately 32% gene knockout and 67% protein reduction in the targeted proto-oncogene polo-like kinase 1 (PLK1). This was sufficient to suppress tumor growth and significantly improved the median survival time of mice bearing orthotopic glioblastoma to 40 days, while inducing negligible side or off-target effects. These results suggest that the developed brain-targeted CRISPR/Cas9 based nanomedicine shows promise for effective human glioblastoma gene therapy.
    Keywords:  Brain-targeting; CRISPR/Cas9; Gene-editing; Glioblastoma; Nanoparticle
  9. Theranostics. 2022 ;12(14): 6422-6436
      Rationale: Messenger RNA (mRNA) vaccine outperforms other kinds of cancer immunotherapy due to its high response rates, easy preparation, and wide applicability, which is considered as one of the most promising forms of next-generation cancer therapies. However, the inherent instability and insufficient protein expression duration of mRNA limit the efficacy and widespread application of the vaccine. Methods: Here, we first tested the possibility of a novel circular RNA (circRNA) platform for protein expression and compare its duration with linear RNA. Then, we developed a lipid nanoparticle (LNP) system for circRNA delivery in vitro and in vivo. Next, the innate and adaptive immune response of circRNA-LNP complex was evaluated in vivo. The anti-tumor efficacy of circRNA-LNP was further confirmed in three tumor models. Finally, the possibility of combination therapy with circRNA-LNP and adoptive cell transfer therapy was further investigated in a late-stage tumor model. Results: We successfully increased the stability of the RNA vaccine by circularizing the linear RNA molecules to form highly stable circRNA molecules which exhibited durable protein expression ability. By encapsulating the antigen-coding circRNA in LNP enabling in vivo expression, we established a novel circRNA vaccine platform, which was capable of triggering robust innate and adaptive immune activation and showed superior anti-tumor efficacy in multiple mouse tumor models. Conclusions: Overall, our circRNA vaccine platform provides a novel prospect for the development of cancer RNA vaccines in a wide range of hard-to-treat malignancies.
    Keywords:  cancer vaccines; circular RNA; hard-to-treat malignancies; lipid nanoparticles; tumor immunotherapy
  10. Pharm Res. 2022 Sep 26.
      INTRODUCTION: Nanoparticle-mediated gene therapy has found substantial clinical impact, primarily focused on lipid-based nanoparticles. In comparison with lipid nanoparticles, polymeric particles may have certain advantages such as increased biocompatibility and controlled release. Our prior studies have found that polymeric mesoscale nanoparticles exhibited specific targeting to the renal proximal tubules. Thus, in this study, we sought to identify formulation parameters that allow for development of polymeric mesoscale nanoparticles encapsulating functional mRNA for delivery into tubular epithelial cells.METHODS: We evaluated particle uptake in vitro prior to exploring formulation parameters related to introduction of a primary mixture of polymer in acetonitrile and hydrophilic mRNA in water. Finally, we evaluated their functionality in a renal tubular epithelial cell line.
    RESULTS: We found that MNPs are endocytosed within 15 min and that the mesoscale nanoparticle formulation procedure was generally robust to introduction of a primary mixture and encapsulation of mRNA. These particles exhibited substantial uptake in renal cells in vitro and rapid (< 1 h) expression of a model mCherry fluorescent protein.
    CONCLUSION: We anticipate these findings having potential in the delivery of specific gene therapies for renal disorders and cancer.
    Keywords:  gene delivery; kidney disease; mRNA; polymeric nanoparticles; translation
  11. Nano Lett. 2022 Sep 28.
      Sepsis is a life-threatening disease caused by systemic bacterial infections, with high morbidity and mortality worldwide. As the standard treatment for sepsis, antibiotic therapy faces the challenge of impaired macrophages and drug-resistant bacteria. In this study, we developed a membrane-camouflaged metal-organic framework (MOF) system for plasmid DNA (pDNA) delivery to combat sepsis. The antimicrobial gene LL37 was efficiently encapsulated in the pH-sensitive MOF, and the nanoparticles were decorated with macrophage membranes in a compatible manner. Macrophage membrane coating allows targeted delivery of LL37 to macrophages and creates macrophage factories for the continuous generation of antimicrobial peptides. Compared to naked nanoparticles, primary bone marrow mesenchymal macrophage membrane-modified nanoparticles greatly improved the survival rate of immunodeficient septic mice through the synergistic effect of efficient gene therapy and inflammatory cytokine sequestration. This study demonstrates an effective membrane biomimetic strategy for efficiently delivering pDNA, offering an excellent option for overcoming sepsis.
    Keywords:  biomimetic nanoparticles; gene therapy; multidrug-resistant bacterial sepsis; pDNA intracellular delivery; primary bone marrow mesenchymal macrophage membrane
  12. ACS Appl Bio Mater. 2022 Sep 28.
      The worldwide steady increase in the number of cancer patients motivates the development of innovative drug delivery systems for combination therapy as an effective clinical modality for cancer treatment. Here, we explored a design concept based on poly(ethylene glycol)-b-poly(2-(dimethylamino)ethyl methacrylate)-b-poly(2-hydroxyethyl methacrylate-formylbenzoic acid) [PEG-b-PDMAEMA-b-P(HEMA-FBA)] for the dual delivery of doxorubicin (DOX) and GTI2040 (an antisense oligonucleotide for ribonucleotide reductase inhibition) to MCF-7 breast cancer cells. PEG-b-PDMAEMA-b-PHEMA, the precursor copolymer, was prepared through chain extensions from a PEG-based macroinitiator via two consecutive atom transfer radical polymerization (ATRP) steps. Then, it was modified at the PHEMA block with 4-formylbenzoic acid (FBA) to install reactive aldehyde moieties. A pH-responsive polymer-drug conjugate (PDC) was obtained by conjugating DOX to the polymer structure via acid-labile imine linkages, and subsequently self-assembled in an aqueous solution to form DOX-loaded self-assembled nanoparticles (DOX-SAN) with a positively charged shell. DOX-SAN condensed readily with negatively charged GTI2040 to form GTI2040/DOX-SAN nanocomplexes. Gel-retardation assay confirmed the affinity between GTI2040 and DOX-SAN. The GTI2040/DOX-SAN nanocomplex at N/P ratio of 30 exhibited a volume-average hydrodynamic size of 136.4 nm and a zeta potential of 21.0 mV. The pH-sensitivity of DOX-SAN was confirmed by the DOX release study based on the significant cumulative DOX release at pH 5.5 relative to pH 7.4. Cellular uptake study demonstrated favorable accumulation of GTI2040/DOX-SAN inside MCF-7 cells compared with free GTI2040/DOX. In vitro cytotoxicity study indicated higher therapeutic efficacy of GTI2040/DOX-SAN relative to DOX-SAN alone because of the downregulation of the R2 protein of ribonucleotide reductase. These outcomes suggest that the self-assembled pH-responsive triblock copolymer is a promising platform for combination therapy, which may be more effective in combating cancer than individual therapies.
    Keywords:  ATRP; GTI2040; cancer therapy; doxorubicin; drug and gene co-delivery; polymer−drug conjugate