A novel λ integrase-mediated seamless vector transgenesis platform for therapeutic protein expression

Abstract

Advances in stem cell engineering, gene therapy and molecular medicine often involve genome engineering at a cellular level. However, functionally large or multi transgene cassette insertion into the human genome still remains a challenge. Current practices such as random transgene integration or targeted endonuclease-based genome editing are suboptimal and might pose safety concerns. Taking this into consideration, we previously developed a transgenesis tool derived from phage λ integrase (Int) that precisely recombines large plasmid DNA into an endogenous sequence found in human Long INterspersed Elements-1 (LINE-1). Despite this advancement, biosafety concerns associated with bacterial components of plasmids, enhanced uptake and efficient transgene expression remained problematic. We therefore further improved and herein report a more superior Int-based transgenesis tool. This novel Int platform allows efficient and easy derivation of sufficient amounts of seamless supercoiled transgene vectors from conventional plasmids via intramolecular recombination as well as subsequent intermolecular site-specific genome integration into LINE-1. Furthermore, we identified certain LINE-1 as preferred insertion sites for Int-mediated seamless vector transgenesis, and showed that targeted anti-CD19 chimeric antigen receptor gene integration achieves high-level sustained transgene expression in human embryonic stem cell clones for potential downstream therapeutic applications.

A non-viral genome editing platform for site-specific insertion of large transgenes

“…An important improvement of our platform was the inclusion of supercoiled seamless target vectors devoid of prokaryotic DNA elements. This was achieved by using Int for in vitro/in vivo site-specific intramolecular recombination between two directly repeated recombination sequences (so-called attachment ( att ) sites) flanking the desired transgene expression cassette in a supercoiled parental substrate vector [ 44 , 45 ]. Thus, besides eliminating unwanted bacterial sequences from the target vector, this approach also reduces the vector size and can enhance transfection efficiency, reduce innate immune responses and contribute to sustained gene expression in human cells [ 33 , 50 – 52 ].…”

“…The integrase-mediated in vitro recombination reaction for seamless vector generation was modified from the method described in [ 45 ]. Briefly, recombination was carried out in a reaction mixture (20 μl) containing 500 ng substrate vector, 10 mM TE buffer, pH 8.0, 150 mM KCl, 57 ng/μl of purified single chain Integration Host Factor (scIHF) [ 46 ] and partially purified Int-h/218 (33.25 ng/μl) [ 43 , 47 ].…”

“…In the context of human genome engineering, none of the SSRs tools has dual capability to produce and subsequently target specific endogenous sequences in the human genome. We previously reported a derivative of the phage lambda integrase (λ-Int) system which is proficient in targeting at endogenous Long INterspersed Elements ( LINE-1 ) in the human genome with seamless vectors [ 43 – 45 ]. The derivative λ-Int system deploys self-sufficient intramolecular recombination to generate seamless vectors and executes specific human genome insertion by subsequent intermolecular recombination [ 44 , 45 ].…”

“…We previously reported a derivative of the phage lambda integrase (λ-Int) system which is proficient in targeting at endogenous Long INterspersed Elements ( LINE-1 ) in the human genome with seamless vectors [ 43 – 45 ]. The derivative λ-Int system deploys self-sufficient intramolecular recombination to generate seamless vectors and executes specific human genome insertion by subsequent intermolecular recombination [ 44 , 45 ]. Using this enhanced strategy, we also demonstrated specific targeting and sustained expression of CD19 chimeric antigen receptors (CARs) in hESCs for potential cancer immunotherapy-related applications [ 45 ].…”

“…The derivative λ-Int system deploys self-sufficient intramolecular recombination to generate seamless vectors and executes specific human genome insertion by subsequent intermolecular recombination [ 44 , 45 ]. Using this enhanced strategy, we also demonstrated specific targeting and sustained expression of CD19 chimeric antigen receptors (CARs) in hESCs for potential cancer immunotherapy-related applications [ 45 ].…”