FEDERICO BRUNELLO
SUPER EVOLVER – CLOSING THE EVOLUTIONARY GAP WITH CANCER. An AI-assisted microfluidics-based fast-tracked evolution platform harnessing CRISPR-Cas13 random RNA mutations and cell-free HITS system for the in-vitro evolution and synthesis of paratope libraries and de-novo enhancement of ADC and mRNAs .
Problem: Immunotherapy is widely adopted as a fourth -line approach against cancer. Due to their inherent genetic instability1, tumours can escape immune response2 and immune therapy3 due to the alteration of epitopes and binding sites exploited as recognition sites by mAbs and ADC-based therapies. Immune therapy customized cells need to be proactively evolved to counter emerging mutations in the tumoral mass to suppress therapeutic escape, which may lead to relapses and, if untreated, metastasis.
*Fig 1 - Use of ADCs in directed cancer therapy*
Issue: Immune therapy long-term effectiveness is hindered by: 1) Composite genotypes and epitopes within tumoral mass 2) Genetic immune escape (GIE) and cancer gene instability 3) Evolutionary selective advantage on surviving cells after mAb and ADCs treatment 4) Emergence of novel mutations causes epitope-specific immune therapy to be ineffective or blind against emerging mutations. It is needed to find a rapidly evolve mAbs and ADCs paratopes to match and follow the evolution of the tumoral mass therapy-resistant cells.
Main goal: to design a parallelized multi-chamber microfluidic system for the rapid evolution or de-novo synthesis of novel arrays of fluorescent protein-barcoded paratopes, starting from de-novo synthesized RNA and/or reverse translation transcripts or paratope structures of existing therapies, to provide a dynamic and flexible tool to fight all possible mutations in the cancer cell.
After entering the microfluidic device as ad cDNA, sequences first transcribed by a cell free TX/RX system mutated in a random but precise way by downstream CRISPR-CAS13 (mutant paratopes mRNA sequences are added with a fluorescent protein tag as GFP and then translated in vitro via the HITS TX/RX in vitro system for eukaryotic cels), with a fluorescent protein, ability to obtain precision editing and robust cleavage for mRNA
Synthesized paratopes are then assembled onto a qubit/cytotoxic/radionuclide-armed iGG heavy chain. mutations ()and the use of a high processivity generate a rapid evolution system to be tested in forced circulation onto a downstream cell miniaturized bioreactor, where a tumor biopsy is ADCs or mABs to follow the rapid evolution of tumoral cells.
Mutated derived cancer mass. Resident CRISPR-Cas13 is random mRNA mutation generations coupled to a downstream RNA barcoding system and a HITS in vitro translation system for fast evolution and testing paratopes. to be assembled onto qubit-tagged Igg. The de-novo generated pools of random mutated laser-printed mRNA sequences based on an existing immunotherapeutic antigen biding site (paratope) are randomly mutated via a CRISPR-Cas13 system a microfluidics system based on TZ
Appoach: 1) AI-generated mRNA leveraged to select and generate mRNA sequences TX/RX, and a palette of to undertake in parallel random mutations of existing ADP-binding paratopes to accelerate the de novo synthesis of paratopes to be dynamically tested on cancer cells.
Goals: To design a multi-channel microfluidics system for the rapid evolution of paratopes from existing ADCs by embeding: a) ribozymes to convert existing ADC epitopes à mRNA b) use of CRISPR-Cas13 system to generate random mRNA mutations c) fusing of RNA with a fluorescent protein palette d) embed a cell-free HITS translation system for colour-coded paratopes production.
VISUAL ABSTRACT
STAGE 0 - As a first approach we are going to take an existing biological drug (e.g. Bevacizumab) based cancer drug being currently used on the patient for immune therapy.
Stage 1: the paratope sequence is in vitro transcribed with a microfluidics kit.
Transcript is subsequently transmitted