Mutation Analysis in Haemophilia A and Investigation of a Keratinocyte Based Gene Therapy for Haemophilia B

Haemophilia A and B are X-linked recessive bleeding disorders caused by a deficiency of the procoagulant function of clotting factors VIII and IX in the intrinsic coagulation pathway, respectively. The two main objectives of this project were: (I) to carry out a comprehensive search for mutations in the factor VIII gene of patients with haemophilia A and (II) to investigate the possibility of keratinocyte-specific expression of factor IX as a potential method for somatic cell gene therapy for haemophilia B, Using a combination of RT-PCR of ectopic factor VIII transcripts and DNA-PCR of individual exons, the entire essential sequence of the factor VIII gene was screened for mutations using chemical cleavage of mismatches (CCM) and single strand conformation analysis (SSCP). A total of 11 different mutations were characterised. These include 7 single base substitutions, 3 deletions and 1 gross mRNA abnormality due to the intron 22- mediated inversion. The 7 single base substitutions included 5 missense (D56E, V162M, G701D, A1834T and R1869I), 1 nonsense (R-5X) and 1 splice donor site mutation in intron 6 (787+3A→G). The 3 deletions included a single base deletion in exon 17 (5697delC), a five base pair deletion in exon 4 (519delTACCT) and a gross deletion of exon 16. Six mutations are predicted to result in a truncated or shortened factor VIII polypeptide. The five missense mutations were evaluated for pathogenicity by analysing the mutated amino acid positions for evolutionary conservation and their involvement with functionally important sequence motifs. Analysis of the sequence in the immediate vicinity of the various mutations revealed sequence features that may have been involved in the mechanism of mutagenesis. To investigate the role of keratinocytes as a target cell type for somatic gene tlierapy of haemophilia B, a number of factor IX (FIX) gene expression vectors were constructed in order to achieve keratinocyte-specific expression from the bovine counterpart of the human cytokeratin 10 (KIO) gene promoter. To optimise the efficiency of recombinant factor IX expression by the basic KIO-FIX-II construct, two sequence modifications were performed. A favourable sequence context was created surrounding the translation initiation codon (K10-FIX-G4) and synthetic oligonucleotides were used to lengthen the 5' leader sequence thus closely resembling the endogenous KIO gene (KIORB-FIX and K10RB-FIX-G4). Since primary keratinocytes do not express KIO in monolayer cultures, a spontaneously transformed, non-tumorigenic, human keratinocyte cell line (HaCaT) was used to assess the absolute and relative factor IX expression by the various gene constructs. It is known that 5-10% of HaCaT cells in confluent cultures express KIO at low levels. Stably transfected HaCaT cell lines (pools and individual clones) were generated with each gene expression construct. Expression of factor IX mRNA was detected in every transfected keratinocyte cell line using RT-PCR analysis. Northern blot analysis of total cellular RNA extracted from all pools of transfected clones (each representing 125-750 clones) detected a 2.5 kb recombinant factor IX transcript. Factor IX protein that was gamma-carboxylated and secreted into conditioned medium, was detected using a double antibody (A5/A7) ELISA. A maximum expression of 35 ng / 10e6 cells / 24 hours was achieved by the K10-FIX-G4 construct. Tlie three constructs with potentially favourable sequence manipulations, expressed significantly liigher amounts of factor IX than the basic KIO-FIX-II construct. As only a few HaCaT cells express KIO, the observed level of factor IX expression can be considered comparable to that achieved by other investigators who used strong viral promoters in similar experiments. These results indicate the suitability of using cytokeratin gene promoters to target the expression of biologically active factor IX in keratinocytes, for somatic gene therapy of haemophilia B