A Bioresponsive Genetically Encoded Antimicrobial Crystal for the Oral Treatment of Helicobacter Pylori Infection.

Helicobacter pylori (H. pylori) causes infection in the stomach and is a major factor for gastric carcinogenesis. The application of antimicrobial peptides (AMPs) as an alternative treatment to traditional antibiotics is limited by their facile degradation in the stomach, their poor penetration of the gastric mucosa, and the cost of peptide production. Here, the design and characterization of a genetically encoded H. pylori-responsive microbicidal protein crystal Cry3Aa-MIIA-AMP-P17 is described. This designed crystal exhibits preferential binding to H. pylori, and when activated, promotes the targeted release of the AMP at the H. pylori infection site. Significantly, when the activated Cry3Aa-MIIA-AMP-P17 crystals are orally delivered to infected mice, the Cry3Aa crystal framework protects its cargo AMP against degradation, resulting in enhanced in vivo efficacy against H. pylori infection. Notably, in contrast to antibiotics, treatment with the activated crystals results in minimal perturbation of the mouse gut microbiota. These results demonstrate that engineered Cry3Aa crystals can serve as an effective platform for the oral delivery of therapeutic peptides to treat gastrointestinal diseases.

Amine-Linked Flavonoids as Agents Against Cutaneous Leishmaniasis.

We have designed, synthesized, and characterized a library of 38 novel flavonoid compounds linked with amines. Some of these amine-linked flavonoids have potent in vitro activity against parasites that cause cutaneous leishmaniasis, a tropical disease endemic in 80 countries worldwide. The most promising candidate, FM09h, was highly active with IC50 of 0.3 µM against L. amazonensis, L. tropica and L. braziliensis amastigotes. It was metabolically stable (39% and 66% of FM09h remaining after 30-minute incubation with human and rat liver microsomes respectively). In L. amazonensis LV78 cutaneous leishmaniasis mouse model, intralesional injection of FM09h (10 mg/kg, once every 4 days for 8 times) demonstrated promising effect in reducing the footpad lesion thickness by 72%, displaying an efficacy comparable to SSG (63%).

Restoration of p53 activity via intracellular protein delivery sensitizes triple negative breast cancer to anti-PD-1 immunotherapy.

BACKGROUND: Although immune checkpoint inhibitors (ICIs) have been shown to yield promising therapeutic outcomes in a small subset of patients with triple negative breast cancer (TNBC), the majority of patients either do not respond or subsequently develop resistance. Recent studies have revealed the critical role of TP53 gene in cancer immunology. Loss or mutation of p53 in cancer cells has been found to promote their immune escape. Given the high mutation frequency of TP53 in TNBC cells, restoration of p53 function could be a potential strategy to overcome their resistance to anti-programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) therapy. Herein, we have assessed the use of Pos3Aa crystal-based platform to mediate the intracellular delivery of p53 protein to restore p53 activity in p53 null tumors and consequently augment anti-PD-1 activity. METHODS: The efficiency of Pos3Aa-p53 crystals in delivering p53 protein was evaluated using confocal imaging, immunofluorescence staining, flow cytometry and RNA-seq. The ability of Pos3Aa-p53 crystals to remodel tumor microenvironment was investigated by examining the markers of immunogenic cell death (ICD) and the expression of PD-L1, indoleamine 2,3-dioxygenase 1, tryptophan 2,3-dioxygenase 2 and type I interferon (IFN). Finally, both unilateral and bilateral 4T1 tumor mouse models were utilized to assess the efficacy of Pos3Aa-p53 crystal-mediated p53 restoration in enhancing the antitumor activity of ICIs. T cells in tumor tissues and spleens were analyzed, and the in vivo biosafety of the Pos3Aa-p53 crystal/anti-PD-1 antibody combination was also evaluated. RESULTS: Delivery of p53 protein into p53-null TNBC 4T1 cells via Pos3Aa-p53 crystals restored the p53 activity, and therefore led to the induction of ICD, activation of type I IFN signaling and upregulation of PD-L1 expression. Pos3Aa-p53 crystals significantly enhanced T cell infiltration and activation in 4T1 tumors, thereby sensitizing them to anti-PD-1 therapy. The combination of Pos3Aa-p53 crystals with anti-PD-1 antibody also induced a systemic antitumor immunity resulting in the inhibition of distal tumor growth with minimal toxicity. CONCLUSION: This study validates that p53 restoration can be an effective approach to overcome ICI resistance and demonstrates that intracellular delivery of p53 protein can be an efficient, safe and potentially universal strategy to restore p53 activity in tumors carrying TP53 mutation.

Cytosolic delivery of CDK4/6 inhibitor p16 protein using engineered protein crystals for cancer therapy.

The tumor suppressor p16 protein is an endogenous CDK4/6 inhibitor. Inactivation of its encoding gene is found in nearly half of human cancers. Restoration of p16 function via adenovirus-based gene delivery has been shown to be effective in suppressing aberrant cell growth in many types of cancer, however, the potential risk of insertional mutagenesis in genomic DNA remains a major concern. Thus, there has been great interest in developing efficient strategies to directly deliver proteins into cells as an alternative that can avoid such safety concerns while achieving a comparable therapeutic effect. Nevertheless, intracellular delivery of protein therapeutics remains a challenge. Our group has recently developed a protein delivery platform based on an engineered Pos3Aa protein that forms sub-micrometer-sized crystals in Bacillus thuringiensis cells. In this report, we describe the further development of this platform (Pos3AaTM) via rationally designed site-directed mutagenesis, and its resultant potency for the delivery of cargo proteins into cells. Pos3AaTM-based fusion protein crystals are shown to exhibit improved release of their cargo proteins as demonstrated using a model mCherry protein. Importantly, this Pos3AaTM platform is able to mediate the efficient intracellular delivery of p16 protein with significant endosomal escape, resulting in p16-mediated inhibition of CDK4/6 kinase activity and Rb phosphorylation, and as a consequence, significant cell cycle arrest and cell growth inhibition. These results validate the ability of these improved Pos3AaTM crystals to mediate enhanced cytosolic protein delivery and highlight the potential of using protein therapeutics as selective CDK4/6 inhibitors for cancer therapy. STATEMENT OF SIGNIFICANCE: Cytosolic delivery of bioactive therapeutic proteins capable of eliciting therapeutic benefit remains a significant challenge. We have previously developed a protein delivery platform based on engineered Pos3Aa protein crystals with excellent cell-permeability and endosomal escape properties. In this report, we describe the rational design of an improved Pos3Aa triple mutant (Pos3AaTM) with enhanced cargo release. We demonstrate that Pos3AaTM-mCherry-p16 fusion crystals can efficiently deliver p16 protein, a CDK4/6 inhibitor frequently inactivated in human cancers, into p16-deficient UM-SCC-22A cells, where it promotes significant G1 cell cycle arrest and cell growth inhibition. These results highlight the ability of the Pos3AaTM platform to promote potent cytosolic delivery of protein therapeutics, and the efficacy of p16 protein delivery as an effective strategy for treating cancer.

Efficient intracellular delivery of p53 protein by engineered protein crystals restores tumor suppressing function in vivo.

Direct delivery of proteins into cells holds significant potential for basic research and drug development. However, the poor endosomal escape of conventional delivery strategies remains a challenge, thus limiting the clinical translation of many protein therapeutics. Herein, we report that engineered Cry3Aa protein (Pos3Aa) crystals formed naturally within Bacillus thuringiensis can serve as a vehicle for efficient cytosolic delivery of bioactive proteins. We showed that Pos3Aa-mediated delivery of tumor suppressor p53 protein, a promising therapeutic candidate found to be inactivated in nearly half of human cancers, resulted in the restoration of p53 function in p53-deficient cancer cells, and thereby sensitized them to 5-fluorouracil chemotherapy as demonstrated in in vitro and in vivo models. Our results validate that Pos3Aa crystals can be a robust and effective platform for the cytosolic delivery of effector proteins, and suggest that efficient uptake and endosomal escape could be critical for efficacious p53 protein-based cancer therapy.

Targeted Myoglobin Delivery as a Strategy for Enhancing the Sensitivity of Hypoxic Cancer Cells to Radiation.

The effectiveness of cancer radiotherapy is frequently hindered by the hypoxia of the tumor microenvironment. Direct delivery of oxygen to hypoxic tumor tissues is an attractive strategy to overcome this hypoxia-associated radioresistance. Herein, we report the generation of submicron-sized particles comprising myoglobin fused to the crystal-forming domain of Cry3Aa protein for the targeted delivery of oxygen to cancer cells. We demonstrate that myoglobin-containing particles were successfully produced in Bacillus thuringiensis with the assistance of the Cry3Aa domain I. Furthermore, these particles could be genetically modified to incorporate the cell penetrating peptide TAT and cell targeting peptide A549.1, resulting in particles that exhibited improved cellular uptake and targeting toward A549 cells. Notably, these myoglobin-containing particles increased the intracellular oxygen levels of A549 cells and thereby sensitized them to radiation. These findings suggest that the targeted delivery of O2-bound myoglobin could be an effective approach to enhance the efficacy of radiotherapy.

In Vivo Enzyme Entrapment in a Protein Crystal.

Cry3Aa is a protein that forms crystals naturally in the bacterium Bacillus thuringiensis. Here we report that coexpression of Cry3Aa and a Proteus mirabilis lipase without recombinant fusion results in the efficient passive entrapment of the lipase within the nanoporous channels of the resulting crystals. This Cry3Aa crystal-mediated entrapment provides multiple benefits to the lipase including a high enzyme loading, significantly improved thermostability, increased proteolytic resistance, and the ability to be utilized as a recyclable biodiesel catalyst. These characteristics, along with its greatly simplified method of isolation, highlight the potential of Cry3Aa crystal-mediated enzyme entrapment for use in biocatalysis and other biotechnological applications.

Targeted delivery of antimicrobial peptide by Cry protein crystal to treat intramacrophage infection.

Antimicrobial peptides (AMPs) have recently attracted great attention due to their rapid action, broad spectrum of activity, and low propensity of resistance development. The successful application of AMPs in the treatment of intracellular infections, however, remains a challenge because of their low penetration efficiency into the pathogen's intracellular niche. Herein, we report that sub-micrometer-sized crystals of the protein Cry3Aa formed within Bacillus thuringiensis are readily and specifically taken up by macrophages. We demonstrate that these protein crystals efficiently encapsulate a known antileishmanial peptide, dermaseptin S1 (DS1), and thereby promote improved cellular uptake of DS1 and its lysosomal accumulation in macrophages. Notably, this targeted delivery of DS1 results in enhanced in vitro and in vivo antileishmanial activity, as well as reduced toxicity to the host macrophages. These findings suggest that the Cry3Aa crystal can be an effective delivery platform for AMPs to treat intramacrophage infections.

Cell type specific expression of Follistatin-like 1 (Fstl1) in mouse embryonic lung development.

Follistatin like-1 (Fstl1) is a secreted glycoprotein and can be up-regulated by TGF-ß1. To better study the function of Fstl1 in lung development, we examined Fstl1 expression in the developing lung, in a cell type specific manner, using a tamoxifen inducible Fstl1-reporter mouse strain. Our results show that Fstl1 is ubiquitously expressed at saccular stage in the developing lung. At E18.5, Fstl1 expression is robust in most type of mesenchymal cells, including airway smooth muscle cells surrounding airways, vascular smooth muscle cells, endothelial cells, and vascular pericytes from blood vessel, but not PDGFRα+ fibroblasts in the distal alveolar sacs. Meanwhile, relative weak and sporadic signals of Fstl1 expression are observed in epithelium, including a subgroup of club cells in proximal airways and a few type II alveolar epithelial cells in distal airways. Our data help to understand the critical role of Fstl1 in lung development and lung disease pathogenesis.