bims-novged 2021-05-09 papers

on Non-viral vectors for gene delivery

Issue of 2021–05–09
nine papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

DNA-inspired nanomaterials for enhanced endosomal escape.
Investigation of water-insoluble hydrophobic polyethylenimines as RNAi vehicles in chronic myeloid leukemia therapy.
Tyrosine-modified linear PEIs for highly efficacious and biocompatible siRNA delivery in vitro and in vivo.
Enhanced siRNA Delivery and Selective Apoptosis Induction in H1299 Cancer Cells by Layer-by-Layer-Assembled Se Nanocomplexes: Toward More Efficient Cancer Therapy.
A novel gene therapy for neurodegenerative Lafora disease via EPM2A-loaded DLinDMA lipoplexes.
α-Tocopherol-anchored gemini lipids with delocalizable cationic head groups: the effect of spacer length on DNA compaction and transfection properties.
Endonasal CNS Delivery System for Blood-Brain Barrier Impermeant Therapeutic Oligonucleotides Using Heterotopic Mucosal Engrafting.
A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications.
Apoptosis induction by siRNA targeting integrin-β1 and regorafenib/DDAB-mPEG-PCL hybrid nanoparticles in regorafenib-resistant colon cancer cells.

Proc Natl Acad Sci U S A. 2021 05 11. pii: e2104511118. [Epub ahead of print]118(19):

DNA-inspired nanomaterials for enhanced endosomal escape.

Jinhyung Lee, Ian Sands, Wuxia Zhang, Libo Zhou, Yupeng Chen.

  To realize RNA interference (RNAi) therapeutics, it is necessary to deliver therapeutic RNAs (such as small interfering RNA or siRNA) into cell cytoplasm. A major challenge of RNAi therapeutics is the endosomal entrapment of the delivered siRNA. In this study, we developed a family of delivery vehicles called Janus base nanopieces (NPs). They are rod-shaped nanoparticles formed by bundles of Janus base nanotubes (JBNTs) with RNA cargoes incorporated inside via charge interactions. JBNTs are formed by noncovalent interactions of small molecules consisting of a base component mimicking DNA bases and an amino acid side chain. NPs presented many advantages over conventional delivery materials. NPs efficiently entered cells via macropinocytosis similar to lipid nanoparticles while presenting much better endosomal escape ability than lipid nanoparticles; NPs escaped from endosomes via a "proton sponge" effect similar to cationic polymers while presenting significant lower cytotoxicity compared to polymers and lipids due to their noncovalent structures and DNA-mimicking chemistry. In a proof-of-concept experiment, we have shown that NPs are promising candidates for antiviral delivery applications, which may be used for conditions such as COVID-19 in the future.

Keywords:  DNA nanotechnology; Janus base nanopieces; Janus base nanotubes; RNA delivery; endosomal escape

DOI:  https://doi.org/10.1073/pnas.2104511118
J Biomed Mater Res A. 2021 May 08.

Investigation of water-insoluble hydrophobic polyethylenimines as RNAi vehicles in chronic myeloid leukemia therapy.

Aysha S Ansari, Remant K C, Xiaoyan Jiang, Hasan Uludaǧ.

  The discovery of RNA interference (RNAi) more than two decades ago opened avenues for avant-garde cancer treatments that possess the ability to evade issues hampering current chemotherapeutic strategies, owing to its specific gene sequence-driven mechanism of action. A potent short interfering RNA (siRNA) delivery vehicle designed to overcome physiological barriers is imperative for successful RNAi therapy. For this purpose, this study explored the characteristics and therapeutic efficacy of low-molecular weight (MW) polyethylenimine (PEI) with high cholesterol substitution, yielding water-insoluble polymers, in chronic myeloid leukemia (CML) K562 cells. A strong impact of cholesterol grafting on the physicochemical attributes of the resultant polymers and their corresponding complexes with siRNA was observed, with the siRNA binding capacity of polymers increasing and complex dissociation sensitivity decreasing with increase in cholesterol content of the polymers. The modified polymer complexes were significantly smaller in size and possessed higher cationic charge compared to the parent polymer. The interaction with anionic heparan sulfate preoteoglycans present on the cell surface was significant in cellular uptake of the complexes. The therapeutic efficacy of siRNA/polymer complexes was reflected in their ability to effectively silence the reporter green fluorescent protein gene and endogenous CML oncogene BCR-ABL as well as significantly inhibit colony formation by K562 cells post BCR-ABL silencing. The results of this study demonstrated beneficial effects of high levels of hydrophobic substitution on low MW PEI on their functional performance bestowing them the potential to be potent RNAi agents for CML therapy.

Keywords:  BCR-ABL silencing; RNAi therapy; cholesterol-grafted polyethylenimine; polymer characterization; siRNA delivery

DOI:  https://doi.org/10.1002/jbm.a.37214
Nanomedicine. 2021 Apr 28. pii: S1549-9634(21)00046-0. [Epub ahead of print] 102403

Tyrosine-modified linear PEIs for highly efficacious and biocompatible siRNA delivery in vitro and in vivo.

Michael Karimov, Marion Schulz, Tim Kahl, Sandra Noske, Malgorzata Kubczak, Ines Gockel, René Thieme, Thomas Büch, Anja Reinert, Maksim Ionov, Maria Bryszewska, Heike Franke, Ute Krügel, Alexander Ewe, Achim Aigner.

  Therapeutic gene silencing by RNA interference relies on the safe and efficient in vivo delivery of small interfering RNAs (siRNAs). Polyethylenimines are among the most studied cationic polymers for gene delivery. For several reasons including superior tolerability, small linear PEIs would be preferable over branched PEIs, but they show poor siRNA complexation. Their chemical modification for siRNA formulation has not been extensively explored so far. We generated a set of small linear PEIs bearing tyrosine modifications (LPxY), leading to substantially enhanced siRNA delivery and knockdown efficacy in vitro in various cell lines, including hard-to-transfect cells. The tyrosine-modified linear 10 kDa PEI (LP10Y) is particularly powerful, associated with favorable physicochemical properties and very high biocompatibility. Systemically administered LP10Y/siRNA complexes reveal antitumor effects in mouse xenograft and patient-derived xenograft (PDX) models, and their direct application into the brain achieves therapeutic inhibition of orthotopic glioma xenografts. LP10Y is particularly interesting for therapeutic siRNA delivery.

Keywords:  RNAi in vivo; Therapeutic siRNA delivery; Tumor xenografts and PDX models; Tyrosine-modified linear polyethlyenimines; siRNA transfection

DOI:  https://doi.org/10.1016/j.nano.2021.102403
Front Mol Biosci. 2021 ;8 639184

Enhanced siRNA Delivery and Selective Apoptosis Induction in H1299 Cancer Cells by Layer-by-Layer-Assembled Se Nanocomplexes: Toward More Efficient Cancer Therapy.

Maryam Sharifiaghdam, Elnaz Shaabani, Zeynab Sharifiaghdam, Herlinde De Keersmaecker, Riet De Rycke, Stefaan De Smedt, Reza Faridi-Majidi, Kevin Braeckmans, Juan C Fraire.

  Nanotechnology has made an important contribution to oncology in recent years, especially for drug delivery. While many different nano-delivery systems have been suggested for cancer therapy, selenium nanoparticles (SeNPs) are particularly promising anticancer drug carriers as their core material offers interesting synergistic effects to cancer cells. Se compounds can exert cytotoxic effects by acting as pro-oxidants that alter cellular redox homeostasis, eventually leading to apoptosis induction in many kinds of cancer cells. Herein, we report on the design and synthesis of novel layer-by-layer Se-based nanocomplexes (LBL-Se-NCs) as carriers of small interfering RNA (siRNA) for combined gene silencing and apoptosis induction in cancer cells. The LBL-Se-NCs were prepared using a straightforward electrostatic assembly of siRNA and chitosan (CS) on the solid core of the SeNP. In this study, we started by investigating the colloidal stability and protection of the complexed siRNA. The results show that CS not only functioned as an anchoring layer for siRNA, but also provided colloidal stability for at least 20 days in different media when CS was applied as a third layer. The release study revealed that siRNA remained better associated with LBL-Se-NCs, with only a release of 35% after 7 days, as compared to CS-NCs with a siRNA release of 100% after 48 h, making the LBL nanocarrier an excellent candidate as an off-the-shelf formulation. When applied to H1299 cells, it was found that they can selectively induce around 32% apoptosis, while significantly less apoptosis (5.6%) was induced in NIH/3T3 normal cells. At the same time, they were capable of efficiently inducing siRNA downregulation (35%) without loss of activity 7 days post-synthesis. We conclude that LBL-Se-NCs are promising siRNA carriers with enhanced stability and with a dual mode of action against cancer cells.

Keywords:  advanced drug delivery; apoptosis; cancer therapy; chitosan; nanomedicine; selenium nanoparticle; siRNA delivery

DOI:  https://doi.org/10.3389/fmolb.2021.639184
Nanomedicine (Lond). 2021 May 07.

A novel gene therapy for neurodegenerative Lafora disease via EPM2A-loaded DLinDMA lipoplexes.

Hari Priya Vemana, Aishwarya Saraswat, Shraddha Bhutkar, Ketan Patel, Vikas V Dukhande.

  Aim: To develop novel cationic liposomes as a nonviral gene delivery vector for the treatment of rare diseases, such as Lafora disease - a neurodegenerative epilepsy. Materials & methods: DLinDMA and DOTAP liposomes were formulated and characterized for the delivery of gene encoding laforin and expression of functional protein in HEK293 and neuroblastoma cells. Results: Liposomes with cationic lipids DLinDMA and DOTAP showed good physicochemical characteristics. Nanosized DLinDMA liposomes demonstrated desired transfection efficiency, negligible hemolysis and minimal cytotoxicity. Western blotting confirmed successful expression and glucan phosphatase assay demonstrated the biological activity of laforin. Conclusion: Our study is a novel preclinical effort in formulating cationic lipoplexes containing plasmid DNA for the therapy of rare genetic diseases such as Lafora disease.

Keywords:  DLinDMA liposomes; Lafora disease; gene delivery; gene therapy; laforin; neurodegenerative disorders; rare diseases

DOI:  https://doi.org/10.2217/nnm-2020-0477
Org Biomol Chem. 2021 May 06.

α-Tocopherol-anchored gemini lipids with delocalizable cationic head groups: the effect of spacer length on DNA compaction and transfection properties.

Mallikarjun Gosangi, Venkatesh Ravula, Hithavani Rapaka, Srilakshmi V Patri.

Understanding the role of structural units in cationic lipids used for gene delivery is essential in designing efficient gene delivery vehicles. Herein, we report a systematic structure-activity investigation on the influence of the spacer length on the DNA compaction ability and the transfection properties of gemini lipids with delocalizable cationic head groups. We have synthesized a series of dimeric cationic lipids varying in spacer length. The DNA binding interactions of liposomal formulations were characterized by gel electrophoresis and ethidium bromide (EtBr) exclusion assays. Condensation potentials were optimized and the best results were observed with cationic lipids possessing a 6 methylene spacer (TIM 6). We found that the size of the lipid/DNA complex decreased with the increase in spacer chain length up to a 6 methylene spacer TIM 6 and increased further. We have optimized the dimeric lipid/DOPE molar formulation using the β-galactosidase activity assay and found that the molar ratio of 1 : 1.5 (gemini lipid/DOPE) showed the maximum transfection among all molar ratios. The cellular uptake and co-localization of lipoplexes were observed by cell analysis and imaging using confocal microscopy. The results confirm that the lipoplex derived from lipid TIM 6 and pCMV-bgal/DNA internalizes via cellular endocytosis. The cytotoxicity studies using the MTT assay revealed that all formulations show comparable cell viability to the commercial standard even at higher charge ratios. Overall, the data suggest that the DNA compaction ability of these lipid dimers depends on the spacer chain length and the gemini lipid containing a six methylene aliphatic spacer has the maximum potential to deliver genes.

DOI: https://doi.org/10.1039/d1ob00475a
Front Pharmacol. 2021 ;12 660841

Endonasal CNS Delivery System for Blood-Brain Barrier Impermeant Therapeutic Oligonucleotides Using Heterotopic Mucosal Engrafting.

Grishma Pawar, Neha N Parayath, Aditya A Sharma, Carlos Coito, Olga Khorkova, Jane Hsiao, William T Curry, Mansoor M Amiji, Benjamin S Bleier.

  The most significant obstacle in the treatment of neurological disorders is the blood-brain barrier (BBB), which prevents 98% of all potential neuropharmaceuticals from reaching the central nervous system (CNS). Brain derived neurotrophic factor (BDNF) is one of the most intensely studied targets in Parkinson's disease (PD) as it can reverse disease progression. BDNF AntagoNAT's (ATs) are synthetic oligonucleotide-like compounds capable of upregulating endogenous BDNF expression. Despite the significant promise of BDNF AT therapies for PD, they cannot cross the blood-brain barrier (BBB). Our group has developed an innovative endonasal heterotopic mucosal grafting technique to provide a permanent method of permeabilizing the BBB. This method is based on established endoscopic surgical procedures currently used in routine clinical practice. Our overall goal for the study was to investigate the distribution and efficacy of BDNF AT's using an extra-cranial graft model in naïve rats using the innovative heterotopic mucosal engrafting technique. BDNF AT cationic liposomes (ideal size range 200-250 nm) were developed and characterized to enhance the delivery to rat brain. Uptake, distribution and transfection efficiency of BDNF AntagoNAT's in saline and liposomes were evaluated qualitatively (microscopy) and quantitatively (ELISA and AT hybridization assays) in RT4-D6P2T rat schwannoma cells and in naïve rats. In vivo therapeutic efficacy of BDNF AT's encapsulated in liposomes was evaluated in a 6-OHDA toxin model of PD using western blot and tyrosine hydroxylase immunohistochemistry. Using complimentary in vitro and in vivo techniques, our results demonstrate that grafts are capable of delivering therapeutic levels of BDNF ATs in liposomes and saline formulation throughout the brain resulting in significant BDNF upregulation in key end target regions relevant to PD. BDNF AT liposomes resulted in a better distribution in rat brain as compared to saline control. The delivered BDNF AT's encapsulated in liposomes also conferred a neuroprotective effect in a rat 6-OHDA model of PD. As a platform technique, these results further suggest that this approach may be utilized to deliver other BBB impermeant oligonucleotide-based therapeutics thereby opening the door to additional treatment options for CNS disease.

Keywords:  antisense oligonucelotides; blood-brain barrier; brain derived neurotrophic factor; central nervous system; liposomes; natural antisense transcript

DOI:  https://doi.org/10.3389/fphar.2021.660841
Cytotherapy. 2021 Apr 30. pii: S1465-3249(21)00184-5. [Epub ahead of print]

A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications.

Heather Kavanagh, Susan Dunne, Darren S Martin, Emily McFadden, Louise Gallagher, Jessica Schwaber, Siobhán Leonard, Shirley O'Dea.

   BACKGROUND AIMS: Next-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications.
METHODS: The Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a real-time cellular impedance assay and a Raji-luciferase mouse tumor model, respectively.
RESULTS: Efficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CAR T cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo.
CONCLUSIONS: The Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary human immune cells with retention of high cell viability and functionality.

Keywords:  CAR T; CD19 CAR; CRISPR; Solupore; TRAC; non-viral delivery

DOI:  https://doi.org/10.1016/j.jcyt.2021.03.002
Am J Cancer Res. 2021 ;11(4): 1170-1184

Apoptosis induction by siRNA targeting integrin-β1 and regorafenib/DDAB-mPEG-PCL hybrid nanoparticles in regorafenib-resistant colon cancer cells.

Mina Zhiani, Mir Ali Mousavi, Kobra Rostamizadeh, Reza Pirizadeh, Abdolreza Osali, Afsaneh Mennati, Behrouz Motlagh, Mojtaba Fathi.

Colorectal cancer (CRC) is regarded as the third most common cancer worldwide. Although Regorafenib as a receptor tyrosine kinase inhibitor (RTKI) disrupts tumor growth and angiogenesis in metastatic CRC (mCRC) patients, drug resistance leads to poor prognosis and survival. Integrin-β1 overexpression has been proposed to be the major player in this regard. Herein, the Regorafenib-resistant human colon cancer cell line (SW-48) was induced, and the Integrin-β1 gene expression, as well as apoptosis, was assessed through the combination of small interfering RNA (siRNA) targeting Integrin-β1 and Regorafenib/Dimethyldioctadecylammonium bromide (DDAB)-methoxy poly (ethylene glycol) (mPEG)-poly-ε-caprolactone (PCL) hybrid nanoparticles (HNPs). In the current study, Regorafenib-resistant SW-48 cell line was generated in which the Regorafenib half-maximal inhibitory concentration (IC50) for non-resistant and resistant cells was 13.5±1.5 µM and 55.1±0.8 µM, respectively. The results of DLS also demonstrated that the size and the charge of the HNPs were equal to 66.56±0.5 nm and +29.5±1.2 mv, respectively. In addition, the Integrin-β1 gene expression was significantly higher in resistant cells than in non-resistant ones (P<0.05). The siRNA/HNP complexes in combination with Regorafenib/HNPs were accordingly identified as the most effective treatment to decrease the Integrin-β1 gene expression and to enhance the apoptosis rate in resistant cells (P<0.001). Overall, the study indicated that combination therapy using siRNA/HNP and Regorafenib/HNPs complex could down-regulate the Integrin-β1 gene expression and consequently trigger apoptosis, and this may potentially induce drug sensitivity.

Keywords: Colorectal cancer; SiRNA; apoptosis; integrin-β1; lipid-polymer hybrid nanoparticle; regorafenib