RNA-based tools for nuclear reprogramming and lineage-conversion: towards clinical applications

Abstract

The therapeutic potential of induced pluripotent stem cells (iPSCs) is well established. Safety concerns remain, however, and these have driven considerable efforts aimed at avoiding host genome alteration during the reprogramming process. At present, the tools used to generate human iPSCs include (1) DNA-based integrative and non-integrative methods and (2) DNA-free reprogramming technologies, including RNA-based approaches. Because of their combined efficiency and safety characteristics, RNA-based methods have emerged as the most promising tool for future iPSC-based regenerative medicine applications. Here, I will discuss novel recent advances in reprogramming technology, especially those utilizing the Sendai virus (SeV) and synthetic modified mRNA. In the future, these technologies may find utility in iPSC reprogramming for cellular lineage-conversion, and its subsequent use in cell-based therapies.

Fig. 1

Efficiency versus safety to estimate reprogrammed cells future for clinical applications. Initially, reprogramming factors were delivered using DNA-integrative reprogramming methods. So far, these methods include (1) retrovirus, (2) lentivirus, including (3) Cre-loxP-mediated transgene excisable variants, and (4) transposons. These are followed by non-integrative DNA-based tools such as (5) adenovirus, (6) self-replicating episomal and (7) standard plasmids, and (8) minicircles. Finally, DNA-free approaches in nuclear reprogramming had been developed, such as (9) protein transduction and of particular success are the RNA-based tools like (10) the Sendai virus (SeV) and (11) synthetic modified mRNA (modRNA)

Fig. 2

Sendai virus (SeV) vector structure and recombinant infective particle generation. a Schematic representation of wild-type RNA SeV genomic organization. Infectively competent SeV-based vectors from first generation express genes of interest (GOI) from a 3′ wild-type SeV vector together with viral proteins NP, P, M, F, HN, and L (see text for details). Second-generation SeV vectors (infectively incompetent), include a T7 promoter and carry an F-defective SeV gene and termosensitive replicative proteins L* and P* genes. b Schematic representation of the two-step procedure for recovery of the F defective SeV vector. In the first step, the functional RNPs are recovered in LLC-MK2 or HEK293Tcells by expressing the viral proteins L, P, and NP together with T7 RNA polymerase. In the second step, RNPs are introduced via a cationic liposome to F-expressing LLC-MK2 cells (LLC-MK2/F7) to produce infectious F-defective virions

Fig. 3

DNA template structure and key molecules used during modified mRNA (modRNA) synthesis. Linear in vitro transcription (IVT) templates incorporate a T7 promoter, 5′ and 3′ untranslated regions (UTRs) flanking an insertion site designed to accept a gene of interest (GOI) and a poly (A) tail. Modified capping molecule and nucleotides structure used for synthesis are also shown: anti reverse cap analog (ARCA) 3′-O-Me-m⁷G(5′)ppp(5′)G, pseudouridine-5'-triphosphate (Pseudo-UTP) and 5-methylcytidine-5′-triphosphate (5-Methyl-CTP)

Fig. 4

Pathways to generate specific differentiated lineages from specific somatic cells. Fully differentiated target cells can be induced by three conceptually separate mechanisms: reprogramming (1) by ectopic expression of Yamanaka factors to induce a pluripotent state which can be later differentiated towards all lineages, (2) by indirect lineage-conversion where an activation phase is required to generate precursor-like cells, or (3) by direct lineage conversion with forced expression of lineage-specific transcription factors. Epigenetic modifications during indirect lineage-conversion are milder compared with the reprogramming process where all epigenetic marks are erased, in contrast to direct conversion which does not imply epigenetic modification