bims-novged 2022-01-09 papers

on Non-viral vectors for gene delivery

Issue of 2022–01–09
nine papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Fine-tuning of polyaspartamide derivatives with alicyclic moieties for systemic mRNA delivery.
A Proton-Activatable DNA-Based Nanosystem Enables Co-Delivery of CRISPR/Cas9 and DNAzyme for Combined Gene Therapy.
ROS-responsive nanoparticle-mediated delivery of CYP2J2 gene for therapeutic angiogenesis in severe hindlimb ischemia.
Robust genome editing in adult vascular endothelium by nanoparticle delivery of CRISPR-Cas9 plasmid DNA.
Precise gene delivery systems with detachable albumin shell remodeling dysfunctional microglia by TREM2 for treatment of Alzheimer's disease.
Controlled Co-delivery of pPDGF-B and pBMP-2 from intraoperatively bioprinted bone constructs improves the repair of calvarial defects in rats.
Direct Cellular Delivery of Exogenous Genetic Material and Protein via Colloidal Nano-Assemblies with Biopolymer.
Enhanced nose-to-brain delivery of siRNA using hyaluronan-enveloped nanomicelles for glioma therapy.
Drug delivery systems for RNA therapeutics.

J Control Release. 2022 Jan 04. pii: S0168-3659(21)00693-3. [Epub ahead of print]

Fine-tuning of polyaspartamide derivatives with alicyclic moieties for systemic mRNA delivery.

Jongmin Yum, Beob Soo Kim, Satomi Ogura, Rimpei Kamegawa, Mitsuru Naito, Yuichi Yamasaki, Hyun Jin Kim, Kanjiro Miyata.

  Development of efficient delivery vehicles for in vitro transcribed mRNA (IVT mRNA) is currently a major challenge in nanomedicines. For systemic mRNA delivery, we developed a series of cationic amphiphilic polyaspartamide derivatives (PAsp(DET/R)s) carrying various alicyclic (R) moieties with diethylenetriamine (DET) in the side chains to form mRNA-loaded polyplexes bearing stability under physiological conditions and possessing endosomal escape functionality. While the size and ζ-potential of polyplexes were comparable among various PAsp(DET/R)s, the transfection efficiencies of polyplexes were considerably varied due to difference in the R moieties of PAsp(DET/R)s and were described by an octanol-water (or buffer at pH 7.3) distribution coefficient (logD7.3). The critical logD7.3 for the efficient in vitro transfection of mRNA was indicated at -2.7 to -1.8. The polyplexes with logD7.3 > -1.8 elicited the much higher in vitro transfection efficiencies. After systemic administration, the polyplexes with logD7.3 from -1.8 to -1.3 elicited the significant mRNA expression specifically in the lungs. The highest mRNA expression in the lungs was achieved by a polyaspartamide derivative having a cyclohexylethyl group (PAsp(DET/CHE)), which induced more than 10-fold increase in mRNA transfection efficiency compared to commercially available lipid nanoparticles. The higher mRNA expression by polyplexes in the lungs was explained well by the preferential lung accumulation of intact mRNA, as determined by quantitative real-time PCR. Our results demonstrate that PAsp(DET/R)s are a promising synthetic material for the enhanced systemic IVT mRNA delivery.

Keywords:  Intravenous administration; Poly(amino acid); Polyplex; mRNA delivery

DOI:  https://doi.org/10.1016/j.jconrel.2021.12.040
Angew Chem Int Ed Engl. 2022 Jan 04.

A Proton-Activatable DNA-Based Nanosystem Enables Co-Delivery of CRISPR/Cas9 and DNAzyme for Combined Gene Therapy.

Feng Li, Nachuan Song, Yuhang Dong, Shuai Li, Linghui Li, Yujie Liu, Zhemian Li, Dayong Yang.

  CRISPR/Cas9 is emerging as a platform for gene therapeutics, and the treatment efficiency is expected to be enhanced by combination with other therapeutic agents. Herein, we report a proton-activatable DNA-based nanosystem that enables co-delivery of Cas9/sgRNA and DNAzyme for the combined gene therapy of cancer. Ultra-long ssDNA chains, which contained the recognition sequences of sgRNA in Cas9/sgRNA, DNAzyme sequence and HhaI enzyme cleavage site, were synthesized as the scaffold of the nanosystem. The DNAzyme cofactor Mn2+ was used to compress DNA chains to form nanoparticles and acid degradable polymer-coated HhaI enzymes were assembled on the surface of nanoparticles. In response to protons in lysosome, the polymer coating was decomposed and HhaI enzyme was consequently exposed to recognize and cut off the cleavage sites, thus triggering the release of Cas9/sgRNA and DNAzyme to regulate gene expressions to achieve a high therapeutic efficacy of breast cancer.

Keywords:  DNA nanotechnology; DNAzyme; Gene editing; Gene therapy; rolling circle amplification

DOI:  https://doi.org/10.1002/anie.202116569
Mater Today Bio. 2022 Jan;13 100192

ROS-responsive nanoparticle-mediated delivery of CYP2J2 gene for therapeutic angiogenesis in severe hindlimb ischemia.

Liang Gui, Youlu Chen, Yongpeng Diao, Zuoguan Chen, Jianwei Duan, Xiaoyu Liang, Huiyang Li, Kaijing Liu, Yuqing Miao, Qing Gao, Zhichao Li, Jing Yang, Yongjun Li.

  With critical limb ischemia (CLI) being a multi-factorial disease, it is becoming evident that gene therapy with a multiple bio-functional growth factor could achieve better therapeutic outcomes. Cytochrome P450 epoxygenase-2J2 (CYP2J2) and its catalytic products epoxyeicosatrienoic acids (EETs) exhibit pleiotropic biological activities, including pro-angiogenic, anti-inflammatory and cardiovascular protective effects, which are considerably beneficial for reversing ischemia and restoring local blood flow in CLI. Here, we designed a nanoparticle-based pcDNA3.1-CYP2J2 plasmid DNA (pDNA) delivery system (nanoparticle/pDNA complex) composed of a novel three-arm star block copolymer (3S-PLGA-po-PEG), which was achieved by conjugating three-armed PLGA to PEG via the peroxalate ester bond. Considering the multiple bio-functions of CYP2J2-EETs and the sensitivity of the peroxalate ester bond to H2O2, this nanoparticle-based gene delivery system is expected to exhibit excellent pro-angiogenic effects while improving the high oxidative stress and inflammatory micro-environment in ischemic hindlimb. Our study reports the first application of CYP2J2 in the field of therapeutic angiogenesis for CLI treatment and our findings demonstrated good biocompatibility, stability and sustained release properties of the CYP2J2 nano-delivery system. In addition, this nanoparticle-based gene delivery system showed high transfection efficiency and efficient VEGF expression in vitro and in vivo. Intramuscular injection of nanoparticle/pDNA complexes into mice with hindlimb ischemia resulted in significant rapid blood flow recovery and improved muscle repair compared to mice treated with naked pDNA. In summary, 3S-PLGA-po-PEG/CYP2J2-pDNA complexes have tremendous potential and provide a practical strategy for the treatment of limb ischemia. Moreover, 3S-PLGA-po-PEG nanoparticles might be useful as a potential non-viral carrier for other gene delivery applications.

Keywords:  CLI, critical limb ischemia; CYP2J2; CYP2J2, cytochrome P450 epoxygenase 2J2; EETs, epoxyeicosatrienoic acids; Gene therapy; Hindlimb ischemia; Nanocarriers; PAD, peripheral artery disease; ROS, reactive oxygen species; Therapeutic angiogenesis

DOI:  https://doi.org/10.1016/j.mtbio.2021.100192
Cell Rep. 2022 Jan 04. pii: S2211-1247(21)01700-9. [Epub ahead of print]38(1): 110196

Robust genome editing in adult vascular endothelium by nanoparticle delivery of CRISPR-Cas9 plasmid DNA.

Xianming Zhang, Hua Jin, Xiaojia Huang, Birendra Chaurasiya, Daoyin Dong, Thomas P Shanley, You-Yang Zhao.

  Vascular endothelium plays a crucial role in vascular homeostasis and tissue fluid balance. To target endothelium for robust genome editing, we developed poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-b-PLGA) copolymer-based nanoparticle formulated with polyethyleneimine. A single i.v. administration of mixture of nanoparticles and plasmid DNA expressing Cas9 controlled by CDH5 promoter and guide RNA (U6 promoter) induced highly efficient genome editing in endothelial cells (ECs) of the vasculatures, including lung, heart, aorta, and peripheral vessels in adult mice. Western blotting and immunofluorescent staining demonstrated an ∼80% decrease of protein expression selectively in ECs, resulting in a phenotype similar to that of genetic knockout mice. Nanoparticle delivery of plasmid DNA could induce genome editing of two genes or genome editing and transgene expression in ECs simultaneously. Thus, nanoparticle delivery of plasmid DNA is a powerful tool to rapidly and efficiently alter expression of gene(s) in ECs for cardiovascular research and potential gene therapy.

Keywords:  CRISPR-Cas9; cardiovascular diseases; endothelial cells; endothelium targeting; gene delivery; gene therapy; genome editing; lung diseases; nanoparticle; non-viral CRISPR delivery

DOI:  https://doi.org/10.1016/j.celrep.2021.110196
Biomaterials. 2021 Dec 30. pii: S0142-9612(21)00716-X. [Epub ahead of print]281 121360

Precise gene delivery systems with detachable albumin shell remodeling dysfunctional microglia by TREM2 for treatment of Alzheimer's disease.

Pengzhen Wang, Peng Yang, Kang Qian, Yixian Li, Shuting Xu, Ran Meng, Qian Guo, Yunlong Cheng, Jinxu Cao, Minjun Xu, Wei Lu, Qizhi Zhang.

  Intervention of the over-activated microglia-aggravated neuroinflammation represents a promising therapeutic strategy for Alzheimer's disease (AD). Upregulation of triggering receptor expressed on myeloid cells-2 (TREM2) attenuates the neuroinflammatory processes and normalizes the dysfunctional microglia. However, Trem2-gene therapy for AD by the effective non-invasive delivery systems is unexploited. Herein, we report the microglia-targeted gene delivery systems (PHSA@PF/pTREM2) composed of a core of fluorinated polyethylenimine condensing the TREM2-encoding plasmid (PF/pTREM2) and a shell of human serum albumin conjugated with both cis-aconitic anhydride and neural cell adhesion molecule-mimetic peptide P2 (PHSA). Thanks to the shedding effect of the albumin coated, PHSA@PF/pTREM2 exhibit prolonged blood circulation and low cytotoxicity. PHSA@PF/pTREM2 achieve brain accumulation as high as 2.17% injected dose per gram of brain and the microglial-targeting effect (targeting specificity of 41.9%) via the systemic administration. The nanocomplexes can be detached PHSA-shell in the acidic endo-lysosomes via the cleavage of cis-aconitic amide bond, resulting in PF/pTREM2 exposure for efficient endo-lysosomal escape and gene transfection. PHSA@PF/pTREM2 upregulate the TREM2 level and regulate microglial polarization toward M2-phenotype for remodeling the inﬂammatory microenvironment and enhanced Aβ clearance, leading to an improvement of cognitive performance in APP/PS1 mice. This work provides a promising gene delivery platform to reverse dysfunctional microglia for AD therapy.

Keywords:  Alzheimer's disease; Gene delivery system; Microglial dysfunction; Microglial targeting; Non-invasive; TREM2

DOI:  https://doi.org/10.1016/j.biomaterials.2021.121360
Biomaterials. 2021 Dec 28. pii: S0142-9612(21)00689-X. [Epub ahead of print]281 121333

Controlled Co-delivery of pPDGF-B and pBMP-2 from intraoperatively bioprinted bone constructs improves the repair of calvarial defects in rats.

Kazim K Moncal, R Seda Tigli Aydın, Kevin P Godzik, Timothy M Acri, Dong N Heo, Elias Rizk, Hwabok Wee, Gregory S Lewis, Aliasger K Salem, Ibrahim T Ozbolat.

  Intraoperative bioprinting (IOB), which refers to the bioprinting process performed on a live subject in a surgical setting, has made it feasible to directly deliver gene-activated matrices into craniomaxillofacial (CMF) defect sites. In this study, we demonstrated a novel approach to overcome the current limitations of traditionally fabricated non-viral gene delivery systems through direct IOB of bone constructs into defect sites. We used a controlled co-delivery release of growth factors from a gene-activated matrix (an osteogenic bioink loaded with plasmid-DNAs (pDNA)) to promote bone repair. The controlled co-delivery approach was achieved from the combination of platelet-derived growth factor-B encoded plasmid-DNA (pPDGF-B) and chitosan-nanoparticle encapsulating pDNA encoded with bone morphogenetic protein-2 (CS-NPs(pBMP2)), which facilitated a burst release of pPDGF-B in 10 days, and a sustained release of pBMP-2 for 5 weeks in vitro. The controlled co-delivery approach was tested for its potential to repair critical-sized rat calvarial defects. The controlled-released pDNAs from the intraoperatively bioprinted bone constructs resulted in ∼40% bone tissue formation and ∼90% bone coverage area at 6 weeks compared to ∼10% new bone tissue and ∼25% total bone coverage area in empty defects. The delivery of growth factors incorporated within the intraoperatively bioprinted constructs could pose as an effective way to enhance bone regeneration in patients with cranial injuries in the future.

Keywords:  Controlled co-delivery; In-situ delivery; Intraoperative bioprinting; Plasmid-DNAs

DOI:  https://doi.org/10.1016/j.biomaterials.2021.121333
ACS Appl Mater Interfaces. 2022 Jan 05.

Direct Cellular Delivery of Exogenous Genetic Material and Protein via Colloidal Nano-Assemblies with Biopolymer.

Ankan Kumar Sarkar, Koushik Debnath, Himali Arora, Pankaj Seth, Nihar R Jana, Nikhil R Jana.

  Direct cytosolic delivery of large biomolecules that bypass the endocytic pathways is a promising strategy for therapeutic applications. Recent works have shown that small-molecule, nanoparticle, and polymer-based carriers can be designed for direct cytosolic delivery. It has been shown that the specific surface chemistry of the carrier, nanoscale assembly between the carrier and cargo molecule, good colloidal stability, and low surface charge of the nano-assembly are critical for non-endocytic uptake processes. Here we report a guanidinium-terminated polyaspartic acid micelle for direct cytosolic delivery of protein and DNA. The polymer delivers the protein/DNA directly to the cytosol by forming a nano-assembly, and it is observed that <200 nm size of colloidal assembly with near-zero surface charge is critical for efficient cytosolic delivery. This work shows the importance of size and colloidal property of the nano-assembly for carrier-based cytosolic delivery of large biomolecules.

Keywords:  direct membrane penetration; gene delivery; guanidinium; nanoparticle; polymer micelle; protein delivery

DOI:  https://doi.org/10.1021/acsami.1c22009
J Control Release. 2021 Dec 30. pii: S0168-3659(21)00687-8. [Epub ahead of print]342 66-80

Enhanced nose-to-brain delivery of siRNA using hyaluronan-enveloped nanomicelles for glioma therapy.

YuLing Yang, XueYan Zhang, SiWen Wu, Rui Zhang, BaiLing Zhou, XiaoYu Zhang, Lin Tang, Yaomei Tian, Ke Men, Li Yang.

  Gliomas are the most malignant brain tumors, and their treatment is very challenging because of the presence of the blood-brain barrier (BBB). Intranasal administration has been considered a noninvasive strategy for glioma therapy in recent years, but our explorations of the intranasal delivery of siRNA-based therapies are still clearly inadequate. In this study, the cell-penetrating peptide DP7-C was enveloped with hyaluronic acid (HA) to develop the multifunctional core-shell structure nanomicelle HA/DP7-C. In vitro studies of HA/DP7-C revealed low cytotoxicity and a higher cell uptake efficiency, which was associated with the interaction between HA and CD44. In vivo experiments indicated that HA/DP7-C delivered the siRNA to the central nervous system through the trigeminal nerve pathway within hours after intranasal administration and that the interaction between HA and CD44 also increased its accumulation at the tumor site. Successful intracellular delivery of an antiglioma siRNA inhibited tumor growth and ultimately prolonged the survival time and decreased the tumor volume in GL261 tumor-bearing mice. In addition, toxicity tests on rats showed no adverse effects on the nasal mucosa and trigeminal nerves. In conclusion, HA/DP7-C is a potential intranasal delivery system for siRNAs in glioma therapy.

Keywords:  DP7-C; Glioma therapy; Nanomicelle; Nose-to-brain; siRNA

DOI:  https://doi.org/10.1016/j.jconrel.2021.12.034
Nat Rev Genet. 2022 Jan 04.

Drug delivery systems for RNA therapeutics.

Kalina Paunovska, David Loughrey, James E Dahlman.

RNA-based gene therapy requires therapeutic RNA to function inside target cells without eliciting unwanted immune responses. RNA can be ferried into cells using non-viral drug delivery systems, which circumvent the limitations of viral delivery vectors. Here, we review the growing number of RNA therapeutic classes, their molecular mechanisms of action, and the design considerations for their respective delivery platforms. We describe polymer-based, lipid-based, and conjugate-based drug delivery systems, differentiating between those that passively and those that actively target specific cell types. Finally, we describe the path from preclinical drug delivery research to clinical approval, highlighting opportunities to improve the efficiency with which new drug delivery systems are discovered.

DOI: https://doi.org/10.1038/s41576-021-00439-4