NFP37 SOMATIC GENE THERAPY Scientific Abstracts

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Prof. Daniel Schümperli;	Zoologisches Institut; Entw.biologie; Universität Bern; Baltzerstr. 4; 3012 Bern; CH; Tel 031 631 46 75; Fax 031 631 41 21; e-mail: SCHUEMPERLI@ZOI.unibe.ch
Title:	A small-RNA expression system for antisense gene therapy.
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SCIENTIFIC ABSTRACT 1997:

We are exploring the potential of the stable U7 small nuclear RNA (snRNA), a factor involved in 3' end processing of animal histone mRNAs, as a delivery system for antisense RNA sequences. The 5' end of U7 RNA which is complementary to part of the processing signal in histone pre-mRNAs can be replaced by antisense sequences to other target RNAs. Such U7 RNA derivatives can either compete for nuclear factors which interact with the same target RNA (splicing factors in particular) or cleave the target RNA via a inserted hammerhead ribozyme or via the his/one-specific processing mechanism. The specific nuclear location is advantageous if early steps in the metabolism of the target RNAs are to be affected.

Our main target for this strategy, so far, has been a type of beta-thalassemic mutations, that affect beta-globin pre-mRNA splicing. Three different mutations located in the second intron of the human beta-globin gene create new 5' splice sites and activate a common cryptic 3' splice site upstream in the same intron. Thus a short intronic sequence carrying an in-frame stop codon is included in the mRNA. Work in the laboratory of our collaborator, Dr. R. Kole, UNC Chapel Hill, has shown that splicing can be reverted to the wild-type pattern by antisense oligonucleotides to these splice sites both in vitro and in a cell culture model. We have now expressed U7 RNA derivatives carrying these antisense sequences in cell lines expressing the mutant 13-globin genes. Using combinations of antisense sequences, we can restore up to 40% correct splicing. We will now begin to adapt this strategy to ex vivo and in vivo situations. Moreover, we will begin to investigate another model target, the RNA genome of human immunodeficiency virus type 1, the causative agent of AIDS.

We are exploring the potential of the stable U7 small nuclear RNA (snRNA), a factor involved in 3' end processing of animal histone mRNAs, as a delivery system for antisense RNA sequences. The 5' end of U7 RNA which is complementary to part of the processing signal in histone pre-mRNAs can be replaced by antisense sequences to other target RNAs. Such U7 RNA derivatives may either compete for nuclear factors which interact with the same target RNA (splicing factors in particular) or cleave the target RNA via a inserted hammerhead ribozyme or via the histonespecific processing mechanism. The specific nuclear location is advantageous if early steps in the metabolism of the target RNAs are to be affected.
Our main target for this strategy, so far, has been a type of beta-thalassemic mutations, that affect beta-globin pre-mRNA splicing. Three different mutations located in the second intron of the human beta-globin gene create new 5' splice sites and activate a common cryptic 3' splice site upstream in the same intron. As a consequence, a piece of the intron with an in-frame stop codon is included in the mRNA and production of functional betaglobin protein is prevented. Antisense U7 constructs targeted against the new 5' and cryptic 3' splice sites induce a stable partial correction of the pre-mRNA splicing pattern and lead to the production of beta-globin protein in tissue culture cells expressing the mutated beta-globin genes. By comparison, we find that the strengths of the different aberrant 5' splice sites influence the accessibility for antisense U7 RNA of the upstream cryptic 3' splice site used in all three mutations, lending support to an exon definition model for pre-mRNA splicing. In addition we have made U7 derivatives capable of binding to two different targets and forcing intron 2 into a looped secondary structure. These double target antisense constructs correct beta-globin pre-mRNA splicing with greatly improved effficiency (>50%) and are active against all three mutations even though in some cases the mutant splice sites are not directly targeted. This strategy provides a potential alternative to gene replacement strategies and holds a strong promise for future clinical applications. We will now begin to adapt this strategy to ex vivo and in vivo situations. Moreover, we have begun to investigate another model target, the RNA genome of human immunodeficiency virus type 1, the causative agent of AIDS.

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