Madaiah Puttaraju

Spliceosome-mediated RNA trans -splicing as a tool for gene therapy

We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the β-subunit of human chorionic gonadotropin gene 6 and pre–trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant β-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.

Partial correction of endogenous ΔF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing

Spliceosome-mediated RNA trans-splicing (SMaRT) was investigated as a means for functionally correcting endogenous ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) transcripts using in vitro human cystic fibrosis (CF) polarized airway epithelia and in vivo human CF bronchial xenografts. Recombinant adenovirus (Ad.CFTR-PTM) encoding a pre-therapeutic molecule (PTM) targeted to CFTR intron 9 corrected transepithelial cyclic AMP (cAMP)-sensitive short-circuit current (Isc) in ΔF508 homozygous epithelia to a level 16% of that observed in normal human bronchial epithelia. Molecular analyses using RT-PCR and western blotting confirmed SMaRT-mediated partial correction of endogenous ΔF508 messenger RNA (mRNA) transcripts and protein. In an in vivo model of ΔF508 CF airway epithelia, human CF bronchial xenografts infected with Ad.CFTR-PTM also demonstrated partial correction of CFTR-mediated Cl− permeability at a level 22% of that seen in non-CF xenografts. These results provide functional evidence for SMaRT-mediated repair of mutant endogenous CFTR mRNA in intact polarized CF airway epithelial models.

Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing

Most messenger RNA precursors (pre-mRNA) undergo cis-splicing in which introns are excised and the adjoining exons from a single pre-mRNA are ligated together to form mature messenger RNA. This reaction is driven by a complex known as the spliceosome. Spliceosomes can also combine sequences from two independently transcribed pre-mRNAs in a process known as trans-splicing. Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology in which RNA pre-therapeutic molecules (PTMs) are designed to recode a specific pre-mRNA by suppressing cis-splicing while enhancing trans-splicing between the PTM and its pre-mRNA target. This study examined the feasibility of SMaRT as a potential therapy for genetic diseases to correct mutations using cystic fibrosis (CF) as an example. We used several versions of a cystic fibrosis transmembrane conductance regulator (CFTR) mini-gene expressing mutant (ΔF508) pre-mRNA targets and tested this against a number of PTMs capable of binding to the CFTR target intron 9 and trans-splicing in the normal coding sequences for exons 10–24 (containing F508). When 293T cells were cotransfected with both constructs, they produced a trans-spliced mRNA in which normal exon 10–24 replaced mutant exon 10. To test whether SMaRT produced mature CFTR protein, proteins were immunoprecipitated from lysates of co- transfected cells and detected by Western blotting and PKA-phosphorylation. Tryptic phosphopeptide mapping confirmed the identity of CFTR. This proof-of-concept study demonstrates that exon replacement by SMaRT can repair an abnormal pre-mRNA associated with a genetic disease.

Development of spliceosome-mediated RNA trans-splicing (SMaRT™) for the correction of inherited skin diseases

Abstract: Gene therapy of large genes (e.g. plectin and collagen genes) is hampered by size limitations for insertions of the currently used viral vectors. To reduce the size of these insertions spliceosome‐mediated RNA trans‐splicing (SMaRT™), which provides intron‐specific gene‐correction at the pre‐RNA level, can be an alternative approach. To test its applicability in skin gene therapy, SMaRT™ was used in the context of the 4003delTC mutation in the collagen XVII gene (COL17A1) causing generalized atrophic benign junctional epidermolysis bullosa. A β‐galactosidase (β‐gal) trans‐splicing assay system was established using intron 51 of COL17A1 as the target for trans‐splicing. In this system, intron 51 is flanked by the 5′exon and the 3′exon of the β‐gal gene, the latter containing two in‐frame stop codons. Cotransfection of a pre‐trans‐splicing molecule consisting of the binding domain of intron 51 and the 3′exon of β‐gal without the stop codons resulted in a 300‐fold increase of β‐gal activity compared to controls. A 2–3‐fold increase in efficiency was obtained through an elongation of the binding domains. Replacement of the complete 3′end of the COL17A1 gene was shown using a collagen XVII mini‐gene construct. The β‐gal assay was used in human keratinocytes to evaluate the influence of a keratinocyte‐specific spliceosome background. Reverse transcription polymerase chain reaction and β‐gal activity assay showed functional correction of the stop‐codons in cultured human keratinocytes and in an immortalized GABEB cell line harbouring the 4003delTC mutation. These results demonstrate that SMaRT™ is feasible in a keratinocyte‐specific context and therefore may be applied in skin gene therapy.

Trans-splicing Into Highly Abundant Albumin Transcripts for Production of Therapeutic Proteins In Vivo

Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy. Here we describe a novel trans-splicing strategy, which targets highly abundant pre-mRNAs, to produce therapeutic proteins in vivo. First, we used a pre-trans-splicing molecule (PTM) that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-mRNA and production of hapoA-I protein. The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice. Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo.