Title: Nanosystems for Gene Editing and Targeted Therapy
Description: Nanomedicine has emerged in the past decades, and a variety of designs for drug/gene delivery have been reported since the concept of nanomedicine was first demonstrated. However, with the exception of a few notable successes, the clinical translation of nanomedicine has been slow. Specificity and delivery efficiency are the major obstacles; only a few nanomedicine systems can effectively reach and release the therapeutic payload at the target site, thereby limiting the therapeutic efficacy. To tackle these issues, this work aims to design new strategies to improve nanomedicine systems at the gene-, protein- and tissue- levels. We applied CRISPR/Cas9 technology for gene targeting. Delivering CRISPR/Cas9 elements, including Cas9 endonuclease and a corresponding guide RNA, allows for specific gene mutagenesis. A conventional gene delivery carrier often has a highly positive charge density for higher transgene expression, but this may result in unfavorable effects on the Cas9 plasmid transfection.

As a large plasmid, strong interaction between the Cas9 plasmid and the polycation with high charge density may hinder the plasmid’s intracellular release. Moreover, high Cas9 expression usually leads to undesirable off-target effects. We addressed these two major obstacles by designing a low-charged density micelle, composed of quaternary ammonium‐terminated poly(propylene oxide) and amphiphilic Pluronic F127. We tested this design on a human papillomavirus (HPV)-induced cervical cancer model to target the HPV oncogene, E7. Our micellar carrier enabled effective Cas9 transfection with a transient Cas9 expression, which offered enhanced Cas9 on-target specificity. This nonviral Cas9‐mediated E7 mutagenesis resulted in significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo. Although CRISPR/Cas9 technology is a powerful toolkit for gene manipulation, gene editing might not be practical for therapeutics in the cancers that develop from endogenous mutations, which may vary among patients and disease stages.

Protein-targeting, therefore, may be a more efficient approach. Aptamer and its selection technology, namely SELEX, offer direct evolution to obtain a nucleic acid ligand that specifically recognizes the protein target. Yet, aptamer screening remains unsatisfactory, and the success rate of SELEX is limited. We designed two approaches to improve the aptamer screening. We first employed a microarray platform to deconvolute the aptamer sequence and identified the aptamer functional motif. The resulted protein-targeting motif with an optimal length and showed enhanced structural and functional characteristics compared with its parental sequence. In addition to sequence optimization, conjunction of two distinct aptamers that recognize different epitopes of the protein target is another approach to improve the aptamer’s affinity. In looking for a rapid way to screen this bivalent aptamer pair, we designed a quantum dot (QD)/ Förster resonance energy transfer (FRET) sensor. Using a thrombin aptamer as a model system, we conjugated an anti-thrombin aptamer with QD and stained the other one with the intercalation dye, YOYO-3.

If the two aptamers recognized different epitopes of thrombin, the conformational change of the two aptamers would take place when interacting with thrombin, and this would induce YOYO-3 dye’s translocation. YOYO-3 would be transferred from the aptamer to QD surface, resulting in a strong FRET signal. In contrast, if they recognized the same epitope, binding competition between two aptamers would inhibit dye translocation, thereby giving a minimal FRET signal. By measuring the FRET signal, we can verify if the two aptamers may form a bivalent pair. Lastly, we integrated mesenchymal stem cell (MSC) with a nanomedicine system to achieve active tissue-targeting. MSC is known to migrate toward certain types of cancer cells by chasing the chemotaxis release from the cancer cells, but the therapeutic payload that MSC can carry is limited. Forming an MSC spheroid allowed the loading of the nanomedicine system with another type of anti-cancer drug. We therefore designed a hybrid MSC/nanomedicine spheroid, which functioned as an active tumor-targeting platform, enabling effective delivery for both cytotoxic protein and chemotherapeutic drugs.

In a heterotopic glioblastoma model, the hybrid spheroid significantly improved the retention of the nanomedicine system at the tumor site, leading to enhanced tumor inhibition in vivo. Collectively, this work demonstrated the effective approaches for gene, protein and tissue targeting by addressing the issues of low specificity and limited delivery efficiency that many current nanomedicine systems face. Particularly, the results may add to the armamentarium of cancer therapeutics, which remains largely challenging and intractable.