In Tools of the Trade, we examined the viral and non-viral
vectors commonly used for
gene delivery. Each of those vectors is designed to deliver normal
copies of a gene into cells that contain only a mutated copy.
There are times, though, when adding a "good" copy of the gene won't solve the problem.
For example, if the mutated gene encodes a
protein that prevents the normal protein from
doing its job, adding back the normal gene won't help. Mutated genes that
function this way are called dominant negative.
How to deal with a dominant negative?
To address this situation, you could either repair the mutated gene's product, or you could
get rid of it altogether. Here are some of the newest methods that scientists are developing as potential approaches to gene therapy.
Each of these techniques also requires a specific and efficient means of delivering the gene to the
A technique for repairing mutations:
The term SMaRT™ stands for "Spliceosome-Mediated RNA Trans-splicing." This technique
targets and repairs the messenger RNA
(mRNA) transcripts copied from the mutated gene. Instead of attempting to replace the entire
gene, this technique repairs just the section of the mRNA transcript that contains the
The sequence of a human gene contains regions that encode the protein
(called exons) and regions that don't encode the protein
After a gene is copied into mRNA, the cell uses RNA-based machinery called
spliceosomes (pronounced SPLICE-oh-zomes)
to cut out the non-coding introns and splice the exons together.
SMaRT™ involves three steps:
- Delivery of an RNA strand that pairs specifically with the intron next to the
mutated segment of mRNA. Once bound, this RNA strand prevents spliceosomes from
including the mutated segment in the final, spliced RNA product.
- Simultaneous delivery of a correct version of the segment to replace the mutated
piece in the final mRNA product
- Translation of the repaired mRNA to produce the normal, functional protein
SMaRT™ is a trademark of Intronn, Inc.
Techniques to prevent the production of a mutated protein:
(pronounced AHL-ih-go-NOOK-leo-tide) gene therapy targets the DNA sequence of a mutated
gene to prevent its transcription.
This technique involves the delivery of short, single-stranded pieces of DNA,
called oligonucleotides, that bind specifically in the groove between the double strands of
the mutated gene's DNA. Binding produces a
triple-helix structure that prevents
that segment of DNA from being transcribed into mRNA.
Antisense gene therapy aims to turn off a mutated gene in a cell by targeting the mRNA
transcripts copied from the gene.
Genes are made up of two paired DNA strands. During transcription, the sequence
of one strand is copied into a single strand of mRNA. This mRNA is called the "sense"
strand because it contains the code that will be read by the cell as it makes a protein.
The opposite strand is the "antisense" strand.
Antisense gene therapy involves the following steps:
- Delivery of an RNA strand containing the antisense code of a mutated gene
- Binding of the antisense RNA strands to the mutated sense mRNA strands,
preventing the mRNA from being translated into a mutated protein
Like antisense, ribozyme (pronounced RYE-bo-ZYME) gene therapy aims to turn off a mutated
gene in a cell by targeting the mRNA transcripts copied from the gene. This approach
prevents the production of the mutated protein.
Ribozymes are RNA molecules that act as enzymes.
Most often, they act as molecular scissors that cut RNA. For example,
spliceosomes (described above) are believed to be a type of ribozyme. Read more about ribozymes and other
forms of RNA in Bringing RNA into View.
Ribozyme gene therapy involves the following steps:
- Delivery of RNA strands engineered to function as ribozymes.
- Specific binding of the ribozyme RNA to mRNA encoded by the mutated gene
- Cleavage of the target mRNA, preventing it from being translated into a protein
Supported by a Science Education Partnership
Award (SEPA) [No. 1 R25 RR16291-01] from the National Center for Research Resources, a component of the
National Institutes of Health, Department of Health and Human Services. The contents provided
here are solely the responsibility of the authors and do not necessarily represent the official
views of NCRR or NIH.
Reviewing the basics of gene expression
To understand how these new approaches to gene therapy work, you will need to know the
basics of gene transcription and translation. For a review, see
How long will it take?
Although the approaches described here are promising, even the most successful are many
years away from becoming standard treatments for genetic disorders.
What does it take to develop a new therapy? Explore the path from the laboratory to the
doctor's office in From Research to Trials.