Different Existing Gene Therapy Approaches
Transcription and Translation Physiology
The DNA sequence of a gene contains a promoter region that is the starting point for the transcription of an antisense DNA strand into pre-messenger RNA (pre-mRNA) by RNA polymerase (Figure 1). This transcription relies on complementary base pairing and pre-mRNA has the same sequence as the sense DNA strand, with T bases replaced by U bases. This pre-mRNA therefore contains both coding regions (exons) and non-coding regions, i.e. intronic regions and untranslated regions (UTR) (Figure 1).
Gene transcription into pre-messenger RNA followed by its splicing and maturation into messenger RNA and lastly translation into a protein. Transcription starts at the gene promoter region. Pre-mRNA contains untranslated sequences: two untranslated regions (UTR), i.e. 5'UTR upstream to the start codon and 3'UTR downstream to the stop codon, and introns in varying numbers. During pre-mRNA splicing, introns are removed to keep only the exons and the two UTR. Maturation leads to the addition of a poly-A tail at the 3' end and a cap at the 5' end; mRNA is then translated, the amino acids sequence of the protein deriving from the codons sequence between start and stop codons in the mRNA.
Pre-mRNA maturation into mature messenger RNA (mRNA) involves splicing, in which the spliceosome enzymatic complex removes introns. The splicing of a pre-mRNA occurs as it is being transcribed. Alternative splicing leads to different protein isoforms. Mature mRNA is then translated into proteins by the ribosomal enzymatic complex, with three nucleic bases or 'codon' coding for a specific amino acid. The protein results from the assembly of all the amino acids coded between the start and stop codons. It can later be subject to post-translational modification, such as cleavage or phosphorylation.
There is growing evidence for a central role of non-coding RNA in health and disease, for instance in neurodevelopment and neurological diseases. These non-coding RNA may be regulatory sequences of the genome, eventually modulating the expression of other genes, and may be a biological signature of specific cell population.[2–6]
Current genetic-based therapies target the different steps preceding—and including—mRNA translation. Various approaches have been described: genome editing, providing a functional copy of the gene without genome integration, avoiding the transcription of the altered gene or favouring the transcription of the wild-type gene in case of heterozygosity, splicing modification of pre-mRNA or downregulation or non-expression of pathologic mRNA.
In vivo genome editing aims at modifying the abnormal gene sequence or integrate a functional copy in living cells. Generation of animal models is greatly facilitated by CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) approach, but genome editing in humans remains much more challenging without full knowledge about off target and long-term effects in long-lasting editing activities.[7–9] Preliminary safety and pharmacodynamic results were published in June 2021 for an ongoing phase 1 clinical study (NCT04601051) using this approach intravenously to treat transthyretin-related hereditary amyloidosis.
With antisense therapies, contrary to genome editing, the genetic repertoire of the target cell remains unchanged and modifications are not passed down to daughter cells. In antisense approaches, as antisense molecules are degraded by the cell, the treatment is reversible and does not permanently affect the cell or the expression of the gene. They can also rely on a stable construct coding for therapeutic antisense transcripts, which are delivered in the target cells using various vectors. These gene constructs are not incorporated into the genome and are not passed down to all daughter cells, but are stable in each cell and can allow for continuous expression of the therapeutic antisense transcripts.
Brain. 2022;145(3):816-831. © 2022 Oxford University Press