Papers, explained in our own words

1. Toward Gene Transfer Nanoparticles as Therapeutics

Erin W. Kavanagh And Jordan J. Green

Viral vectors dominate gene therapy but suffer from immunogenicity, insertional mutagenesis risk, cargo-size limits, and manufacturing challenges. Non-viral nanoparticles are safer, more economical, and easier to scale, but their clinical translation has been limited by inefficient delivery, especially for DNA and gene-editing cargoes that must reach the nucleus. There is a need to review non-viral nanomaterial design, delivery barriers, and. Viral vector dominance: Approximately 70% of gene therapy clinical trials to date have used viruses. - LNP gene editing in liver: A single administration of CRISPR-Cas9 LNPs achieved >97% reduction in target protein.

2. Microfluidic technologies and devices for lipid nanoparticle-based

Maeki M, Uno S, Niwa A, Okada Y, Tokeshi M

RNA-loaded lipid nanoparticles (LNPs) are clinically validated for siRNA and mRNA delivery, but their therapeutic performance depends critically on reproducible control of size, size distribution, zeta potential, and RNA encapsulation efficiency. Microfluidic devices offer precise mixing, rapid ethanol dilution, high reproducibility, high-throughput formulation screening, and continuous production, making them attractive for standardized LNP. Increasing PEG-lipid from 1 to 5 mol% reduced siRNA-LNP size from 54 nm to 28 nm; DLin-KC2-DMA LNPs achieved 50% FVII silencing at 0.01 mg/kg in mice. - Optimized mRNA-LNP formulations improved delivery and expression.

3. Encapsulation of Large-Size Plasmids in PLGA Nanoparticles for Gene Editing: Comparison of Three Different Synthesis Methods

Tresa Lopez-Royo, Victor Sebastian, Laura Moreno-Martinez, Laura Uson, Cristina Yus, Teresa Alejo, Pilar Zaragoza, Rosario Osta, Manuel Arruebo, Raquel Manzano

CRISPR/Cas gene-editing components are often encoded on large plasmids (9–19 kb), which are difficult to encapsulate and transfect. Most PLGA nanoparticle (NP) gene-delivery studies use small nucleic acids (siRNA) or small plasmids (<6 kb), and the structural integrity and functional performance of large plasmids in PLGA NPs remain poorly characterized. There is a need to compare synthesis methods for encapsulating large plasmids without. pDNA stability: Batch ultrasound double emulsion completely degraded the 9.4 kb plasmid (standard) or left only 8.80% supercoiled (modified). Microfluidics-assisted double emulsion avoided degradation but produced only.

4. Nanomedicine-based cancer immunotherapy: recent trends and future perspectives

Cancer immunotherapy has shown efficacy but is limited by serious adverse effects, nonspecific inflammation, and challenges in spatiotemporal control of immune responses. Nanomedicine and functionalized nanosystems offer programmable pharmacokinetics, co-delivery of immunomodulators, and targeted delivery, potentially improving immunotherapy outcomes. Antigen-capturing NPs (AC-NPs) plus αPD-1 improved cure rate by 20% in B16F10 melanoma, expanded CD8⁺ cytotoxic T cells, and increased CD4⁺/Treg and CD8⁺/Treg ratios. - Cationic lipid-assisted PEG-b-PLGA NPs (CLANs).

5. An ionizable lipid toolbox for RNA

Han X, Zhang H, Butowska K, Swingle Kl, Alameh M-G, Weissman D, Mitchell Mj

RNA therapeutics—ASOs, siRNAs, miRNAs, mRNAs, and CRISPR-Cas9 sgRNAs—are limited by nuclease degradation, large size, and negative charge. Ionizable lipids are the key LNP component that condenses RNA, enables endosomal escape, and reduces toxicity. Since 2008, many ionizable lipids have been created, but the field lacks systematic categorization to guide next-generation design. MC3 is used in the FDA-approved siRNA drug Onpattro (patisiran) for hereditary transthyretin amyloidosis. - Optimized C12-200 formulation increased mRNA expression 7-fold vs standard formulation. - 7C1 achieved ~80%.

6. Lipid Nanoparticle-Mediated Delivery of Mrna Therapeutics and Vaccines

Likely Citation: Swingle Kl, Hamilton Ag, Mitchell Mj

mRNA is degraded by nucleases and cannot easily cross cell membranes because of its large size and negative charge. Delivery therefore requires encapsulation in vehicles such as lipid nanoparticles (LNPs) to enable protein replacement, vaccines, and gene-editing applications. Onpattro was the first FDA-approved LNP-nucleic acid therapeutic, for polyneuropathy caused by transthyretin amyloidosis. - Pfizer-BioNTech and Moderna mRNA-LNP COVID-19 vaccines received FDA emergency use authorization.

7. Lipid nanoparticles for mRNA delivery

Hou X, Zaks T, Langer R, Dong Y

mRNA has therapeutic potential for vaccines, protein replacement, cancer immunotherapy, cellular reprogramming, and genome editing, but it requires safe, effective, and stable delivery systems to protect it from degradation and enable cellular uptake and mRNA release. Lipid nanoparticles have entered the clinic for mRNA delivery, most notably in COVID-19 mRNA vaccines. COVID-19 mRNA vaccines mRNA-1273 and BNT162b2 showed ~95% efficacy in phase III trials and use ionizable LNPs (SM-102 and ALC-0315, respectively). - Influenza mRNA-1440: 100 µg dose induced 78.3% HAI and 87.0% MN.

8. Lipid Nanoparticles for Organ-Specific mRNA Therapeutic Delivery

Zak Mm, Zangi L

mRNA therapeutics are limited by innate immune activation, rapid RNase degradation, and inefficient delivery to target organs. Although LNPs are the only clinically approved RNA therapeutic carriers, most systemically delivered LNPs accumulate in the liver, so organ-specific delivery remains a major barrier for protein replacement, cancer immunotherapy, and gene editing. LNPs are the only RNA therapeutic carriers approved for clinical use at the time of the review. - PEG content from 1% to 5% produces LNPs approximately 100 nm to 20 nm in size; 0.5% PEG gave highest subretinal.

9. Lipid- and polymer-based nanoparticle systems for the delivery of CRISPRCas9

Ashok B, Peppas Na, Wechsler Me

CRISPR/Cas9 genome editing holds major therapeutic promise, but in vivo delivery is arguably the most difficult barrier to clinical translation. Efficient, selective transport of editing components to target cells/tissues while minimizing off-target effects remains unresolved. This review critiques lipid- and polymer-based nanoparticle delivery strategies for CRISPR/Cas9. LPO1 lipid nanoparticle: >97% knockdown of mouse TTR protein. - BAMEA-O16B bioreducible LNP: up to 90% GFP knockout in HEK cells; 80% PCSK9 knockdown in mouse serum after IV injection. - Cationic lipid NP with.

10. Polymer-Stabilized Cas9 Nanoparticles and Modified Repair Templates Increase Genome Editing Efficiency

David N. Nguyen, Theodore L. Roth, P. Jonathan Li, Peixin Amy Chen, Ryan Apathy, Murad R. Mamedov, Linda T. Vo, Victoria R. Tobin, Daniel Goodman, Eric Shifrut, Jeffrey A. Bluestone, Jennifer M. Puck, Francis C. Szoka, Alexander Marson

CRISPR-Cas9 genome editing in clinically relevant primary cells is limited by inefficient homology-directed repair (HDR) and cytotoxicity of exogenous DNA. Improvements are needed to increase editing efficiency, cell viability, and generalizability across cell types for adoptive cell therapies and research. tCTS shuttle: Facing orientation of tCTS on both 5′ and 3′ homology arms improved knock-in at TRAC. Representative flow cytometry: dsDNA vs tCTS — 45.7% vs 68.7%, 16.9% vs 54.1%, 18.7% vs 47.3%, 24.1% vs 51.3% across.

11. Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion

Rui Yan Yuan, Mahita Varanasi, Shanelle Mendes, Hannah M. Yamagata, David R. Wilson, Jordan J. Green

CRISPR-Cas9 gene editing requires efficient intracellular delivery of large cargo. Viral vectors have packaging and production limitations, and nonviral CRISPR plasmid delivery had mainly been studied for single-site knockout, not for gene deletion requiring two cleavage events. The transfection requirements for 1-cut vs. 2-cut edits were unclear. Transfection: >80% of cells were transfected in both HEK293T and B16-F10 using GFP reporter. However, geometric mean expression in HEK293T was nearly 1 order of magnitude higher than B16-F10. - 1-cut knockout: Up to 70%.

12. The once and future gene therapy

Karen Bulaklak And Charles A. Gersbach

Gene therapy is at an inflection point: recent approvals and clinical successes have validated the field, but broader application is still limited by immune responses to vectors, inefficient delivery outside the retina and liver, and regulatory frameworks that are poorly suited to personalized, potentially curative therapies. The comment summarizes recent advances and expectations for the near future. Approved therapies: Spinraza (2016), Exondys 51 (2016), Vyondys 53 (2019), Kymriah (2017), Yescarta (2017), Tescartus, Luxturna (2017), Zolgensma (2019). - First CRISPR disease-modifying efficacy: CTX001 for sickle cell.

13. Recent advancements in liposome technology

Nina Filipczak, Jiayi Pan, Satya Siva Kishan Yalamarty, Vladimir P. Torchilin

Liposomes remain an important nano-sized drug delivery system because of their biomimetic bilayer, ease of preparation, and biocompatibility, but clinical translation is still limited by reproducibility, scale-up, stability, and payload-specific barriers. The review updates recent advances in liposome preparation, excipients, administration routes, and novel modalities including nucleic acid therapies, CRISPR/Cas9, and immunotherapies. DOTAP-based liposome delivery of Cas9 and sgRNA plasmid achieved 39% gene-editing efficiency in knocking out a GFP reporter in HEK293 cells. - DOTAP/Chol/DOPE liposomes delivering miR-34b-5p to thyroid carcinoma reduced.

14. Nanoparticles for nucleic acid delivery: Applications in cancer immunotherapy

Mukalel Aj, Riley Rs, Zhang R, Mitchell Mj.

Nucleic acid therapeutics—DNA, mRNA, and CRISPR/Cas9 genome editing systems—hold significant potential for cancer immunotherapy but face major delivery barriers, including rapid in vivo degradation, poor cellular uptake, required nuclear entry, and toxicity in healthy tissues. Nanoparticle delivery systems are being engineered to safely and effectively deliver these therapeutics to immune cells. Ionizable LNPs with CpG ODN co-administered with tumor antigens showed preferential immune cell uptake in lymph nodes and greater tumor rejection in a murine EG7-OVA model. - PLL-coated polystyrene NPs delivering OVA.

15. RNA delivery biomaterials for the treatment of genetic and rare diseases

Zhao W, Hou X, Vick Og, Dong Y.

Genetic and rare diseases (GARDs) affect more than 350 million patients worldwide, and many lack effective treatments. RNA-based therapeutics—siRNA, mRNA, miRNA, lncRNA, and RNA-containing genome editing systems—are promising, but naked RNA has poor cellular penetration, is degraded in biological fluids, and does not accumulate in target organs after systemic administration. Non-viral RNA delivery biomaterials are needed to enable systemic. Patisiran became the first FDA-approved siRNA drug in 2018; LNP delivers siRNA to hepatocytes, targeting TTR for hATTR. - DLin-KC2-DMA SNALPs showed in vivo activity at 0.01 mg/kg in mice and TTR silencing ED50 ~0.3.

16. A Mobile Endocytic Network Connects Clathrin-Independent Receptor Endocytosis to Recycling and Promotes T Cell Activation

Ewoud B. Compeer, Felix Kraus, Manuela Ecker, Gregory Redpath, Mayan Amiezer, Nils Rother, Philip R. Nicovich, Natasha Kapoor-Kaushik, Qiji Deng, Guerric P.B. Samson, Zhengmin Yang, Jieqiong Lou, Michael Carnell, Haig Vartoukan, Katharina Gaus, Jérémie Rossy

Polarized endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation, yet the cellular mechanisms that coordinate internalization of surface receptors with sustained delivery back to the plasma membrane remain incompletely understood. The role of flotillins in defining a clathrin-independent endocytic route and supporting receptor recycling is unclear, particularly in the context of TCR. ### TCR Internalization Dynamics | Finding | Detail | |-------------|------------| | TCR internalization | Constitutive in resting cells; rapidly increased upon activation (most molecules internalized within 20 s) | |.

17. State-of-the-Art Design and Rapid-Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery

Evers Mjw, Kulkarni Ja, Van Der Meel R, Cullis Pr, Vader P, Schiffelers Rm

Free siRNA, mRNA, pDNA, and CRISPR/Cas9 components are rapidly degraded and cleared, so delivery systems are required. LNPs are the most clinically advanced non-viral siRNA carriers; Patisiran reached Phase III and market application in late 2017. The field needed a clearer understanding of LNP design parameters and scalable rapid-mixing production methods. DLin-MC3-DMA has apparent pKa 6.44; LNP formulation at 40/10/40/10 mol% had ED50 0.03 mg/kg, while 50/10/38.5/1.5 mol% had ED50 0.005 mg/kg in mice. - cKK-E12 LNP had ED50 0.002 mg/kg; YSK13-C3 LNP had ED50 0.015 mg/kg.

18. PLGA-Based Nanoparticles in Cancer Treatment

Sima Rezvantalab, Natascha Ingrid Drude, Mostafa Keshavarz Moraveji, Nihan Güvener, Emily Kate Koons, Yang Shi, Twan Lammers, Fabian Kiessling

PLGA-based nanoparticles are widely investigated for cancer therapy, but their clinical translation remains limited. A consolidated overview was needed of how PLGA NP physicochemical properties, biological behavior, targeting strategies, and combination therapies interact—and why promising preclinical results have rarely translated into approved cancer nanomedicines. PLGA molecular weight controls release: At day 18, payload release was 95%, 66%, 50%, and 23% for PLGA Mw of 14.5, 45, 85, and 213 kDa, respectively. PTX-loaded NP sizes increased with Mw: 122 ± 3, 133 ± 2, and 160 ± 2.

19. Non-viral vectors for gene-based therapy

Yin H, Kanasty Rl, Eltoukhy Aa, Vegas Aj, Dorkin Jr, Anderson Dg.

Gene-based therapy has potential to treat a wide range of diseases, but clinical success is limited by delivery barriers. Viral vectors, while efficient, carry risks of carcinogenesis, immunogenicity, broad tropism, limited packaging capacity, and manufacturing difficulties. Non-viral vectors offer improved safety, larger payload capacity, and easier synthesis, but have low delivery efficiency relative to viruses. Advances in material sciences. ALN-PCS02 (LNP-siRNA targeting PCSK9): Phase I trial showed substantial reduction of PCSK9 and LDL cholesterol with no serious adverse effects. - ALN-TTR02 (Patisiran, LNP-siRNA targeting TTR): Phase II/III; DLinDMA.