Intronn -- Technology

Applications

Spliceosome-mediated RNA trans-splicing or SMaRT™ provides an effective means to reprogram mRNAs and the proteins they encode.

Applications: There are a large number of trans-splicing applications, each governed by the nature of the coding sequences included in the pre-trans-splicing molecule (PTM). PTMs that encode correct genetic sequences or toxins have been used as RNA therapies to repair mutant mRNAs from genes associated with human disease or to kill diseased cells (e.g. cancer cells). Because the coding sequence can consist of one or more exons, a single PTM can be used to correct all the mutations in the region of the target RNA that is replaced. PTMs have also been developed that contain reporter genes. These PTMs have been used for real-time imaging of gene expression. Alternatively, PTMs containing mutant sequences or stop codons can be used to reduce wild-type gene expression and may be useful for target validation.

In addition to PTMs with binding domains comprised of sequences complementary to specific pre-mRNA targets, populations of PTMs with randomized binding domain sequences are capable of nonspecific trans-splicing. Such nonspecific trans-splicing can be used to identify alternative splice sites. Additionally, such randomized trans-splicing could be used for molecular evolution by shuffling specific exon(s) in the PTM with other exons expressed by the genome. The resulting library of exon-shuffled proteins could then be selected for recombinant proteins with optimized or modified function.

In Vivo and In Vitro Proof-of-Principle Studies of SMaRT™ Technology

Target mRNA	Size of PTM Insert (kb)	Host System	Positive results at the level -	Investigators
Factor VIII	1.7	I.V. injection into FVIII knock out mice	RNA sequence FVIII activity levels 20% of physiological tail-clip challenge	C. Walsh Mt. Sinai & Intronn
CFTR	3.2	respiratory epithelial human CF specimens xenograft into rodents LacZ transgenic mouse	RNA sequence western blot functional repair in CF polarized epithelia 16% iodide efflux xenograft functional repair – 22%	J. Engelhardt Univ of Iowa & Intronn
Apolipoprotein A1	1.79	cell models	RNA sequence	Intronn
Mouse albumin	0.8	cell models albumin-ApoA1 fusion normal mice	RNA sequence western blot cholesterol efflux	Intronn
Alpha1 antirypsin	0.7	cell models	RNA sequence	Intronn
SMN2 (spinal muscular atrophy)		cell models SMN deficient SMA fibroblasts	RNA SMN expression snRNP assembly	Coady et al, U Missouri
DNA protein kinase (with Sleeping Beauty transposon)	0.6	DNA-PKcs protein production Radiation resistance	RNA Protein production Correction in SCID multipotent adult progenitor cells	Zayed et al, U Minnesota and Intronn
Albumin		Single chain monoclonal antibody vs HPV	RNA Immunoblot Ab production in mice	Intronn
Collagen 17A	0.8	cell models endogenous repair	5' trans-splicing PCR Immunoblot	J.W. Bauer Salzburg , Austria & Intronn
PLEC1		cell models	RNA Sequence 50% trans-splicing	J.W. Bauer Salzburg , Austria & Intronn
HPV (E6 and E7)	1.38 0.24	cell models integrated target xenograft	RNA sequence up to 70% trans-splicing imaging gene expression	C. Baker NCI & Intronn S. Gambhir Stanford & Intronn
Tau	0.53	cell models	RNA Sequence	Jean-Marc Gallo, Kings College London & Intronn
HCG, β subunit	0.6	xenograft into nude mice with diphtheria toxin subunit A	RNA sequence cell killing	Intronn
β-galactosidase	1.38	model system using various cell lines	RNA sequence western blot restoration of function	Intronn
CD40 ligand		cell lines ex vivo bone marrow into KO mice	RNA sequence restoration of function	Tahara et al
Antibody heavy and light chains		B cell lymphoma cell lines	RNA immunology	Schlesinger et al
Cobratoxin segmental trans-splicing		Cell models In vivo models	RNA cell and animal killing	Pergolizzi et al

Explanatory Notes:

Explanatory Notes:
• RNA : RNA data conforming the presence of repaired RNA and quantitation of trans-splicing efficiency by RT-PCR
• Sequence: Sequence data confirming the accuracy of trans-splicing
• Western Blot: Western analysis confirming the expression of full length protein
• Cell killing: Trans-splicing mediated cell killing in cells or in vivo
• Functional repair: Correction of CFTR-mediated Cl- channel activity in CF polarized epithelia and xenografts 16-22%
• Iodide influx: Another measurement of CFTR Cl- channel activity
• snRNP assembly: Small nuclear ribonucleotide assembly, a function mediated by SMN gene
• Trans-splicing: qRT-PCR measurements of trans-splicing efficiency
• Efflux: ABC1 mediated transfer of cellular cholesterol into ApoA1 acceptor
• FVIII levels: Assessment of activity level of circulating FVIII
• Tail clip challenge: Assessment of phenotypic correction of the bleeding defect measured by survival
• Restoration of function: Ability to be immunized with test antigen