RNA-based gene therapy for Haemophilia B

Peppelenbosch MP, Milano F, Ferreira CV, Knapisnka A, Diks SH, Spek A
Source: Curr Genomics
Publication Date: (2005)
Issue: 6(6): 401-4
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
Monocyte, human
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b
2x10^6 T lymphocytes were mixed with 5 µg/ml GFP-DNA and nucleofected using Human T Cell solution, program U14. Monocyte were harvested and transfected using 0,5 up to 2x 10^6 cells, Nucleofector solution V, and 5 µg/ ml of GFP-DNA; in a separated reaction cells were mixed with the Nucleofector solution V and 5 µg/ml of autologous GFP-RNA; program A27 was used. For assessing the efficiency of autologous RNA transfection peripheral blood lymphocytes were transfected with a GFP expression construct, resulting in a transfection efficiency of 60% as assessed by FACS analysis. Subsequently RNA was isolated from these cells and used as a source of RNA for autologous RNA transfection. When 5000 ng/2 million cells was subsequently nucleofected into monocytes and analysed for GFP fluorescence 24 hrs later, it appeared that 78% of the thus transfected monocytes was producing significant amounts of GFP protein. For comparison, monocytes were also transfected with a GFP expression construct. GFP expression after the latter procedure was only slightly higher as that observed after RNA transfection. The proportion of transfected cells when GFP production in RNA-transfected lymphocytes was measured is even significantly better as that observed in monocytes, but the absolute level of GFP per cell is lower. Nevertheless RNA is efficiently expressed by monocytes and lymphocytes after Nucleofection.
Haemophilia B, a deficiency in clotting factor IX (FIX), occurs in about 1 in 25.000 males. Of these patients, approximately 40% are characterized as having 'severe' haemophilia (FIX below 1% of normal). Although the use of plasma-derived or rFIX has extended the lifespan of these patients, they remain afflicted by a variety of sequelae of the disease. Interestingly, a relatively small increase in the levels of FIX results in a dramatic increase in the quality of life, requiring rFIX substitution only prior to dental and surgical procedures. This latter trait of the disease suggests that gene therapy only needs to achieve modest expression and makes this disease a prime candidate for proof-of-concept of gene therapy. Current protocols for gene therapy entail the risk of malignant transformation. In addition the viral vectors may cause substantial pathology. Hence, the field requires the development of inherently safe gene therapy. Recently, developed protocols have been developed to transduce peripheral blood cells with high efficiency using RNA as a vector. These protocols in which the inherently safe RNA-based gene therapy approach will be validated and implemented for haemophilia B will provide in turn providing important proof-of concept for this type of therapy for more prevalent diseases but requires optimization of this strategy, probably by enhancing RNA stability using artificial nucleotides and modified UTRs, enhanced production of protein by codon usage optimization and the use of multi-cistronic constructs, and enhanced secretion of the protein employing target cell tailored secretion signals.