Lentivirus Vectors for Cystic Fibrosis Gene Therapy

Guest Writer (1997):
Pat Metharom, BSc(Hons)
Address:
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston, Queensland, AUSTRALIA

Construction of a Lentivector

To construct a delivery vector, certain essential cis-acting sequences must be retained within the retroviral vector genome. These include:

1. The packaging signal sequence (Y) which ensures the encapsidation of the vector RNA into virions.
2. Elements that are necessary in the reverse transcription process:
   
   
   • Primer binding site (PBS) - which binds the tRNA primer of reverse transcription
   • Terminal repeat (R) sequences - to guide reverse transcriptase between the RNA strands during DNA synthesis
   • Purine-rich region 5' of the 3' LTR - which acts as the priming site for synthesis of the second (+) DNA strand
     
     
3. Specific sequences near the ends of the LTRs that are necessary for the integration of the proviral vector into the chromosome of the host cell. The most common retroviral vector designs use the LTR of the virus backbone and an internal promoter to drive the expression of the foreign gene⁵². This approach gives rise to the phenomenon of "promoter suppression". When selection is applied for one gene from multiple transcription units, the expression of the other gene can be reduced or lost completely.

An internal ribosome entry site (IRES) sequence may be used instead of an internal promoter in a vector with two or more foreign genes to avoid the potential of promoter suppression. An IRES permits multiple proteins to be produced from a single vector without alternative splicing or multiple transcription units, hence increasing the stability of the transferred gene⁵³.

The essential minimum packaging signal (Y) sequence of lentiviruses is still unclear. It is generally accepted to be in a region between the 5' LTR splice donor and the gag start codon.

Addition of 5' gag sequences to the vector backbone has been shown to increase packaging efficiency^54,55. The 3' env gene fragment encompassing the Rev response element (RRE) was reported to enhance the encapsidation of vectors when placed upstream of the heterologous genes^56,57.

Inclusion of this fragment allows accumulation of unspliced vector RNA in the presence of Rev protein and enhances the transduction of recombinant vectors⁵⁶.

By incorporating the above information, the vector design, shown below, has the potential to overcome the current low titer production and transduction efficiency of lentiviral vectors.

Ideally, a recombinant lentiviral vector would be the best gene transfer system for noncycling cells. It has promising clinical applications, especially for cystic fibrosis gene therapy.

Lentivirus-based Vectors for CF Gene Therapy

Lentiviruses are part of the family Retroviridae. Like other retroviruses, they are enveloped viruses that carry a core of RNA encoding their genetic information. The structure of a lentivirus genome is shown in the figure below.

Figure 1. Schematic representation of lentiviral genome. LTR (long terminal repeats) - contains viral promoter and enhancer. SD - splicing donor. Y - packaging signal. Gag - codes for virion core. Pol - generates reverse transcriptase, endonuclease, and protease. Env - codes for envelope protein

Apart from boasting some of the best properties of current retrovirus vectors for gene delivery (see previous section), lentiviruses are the only retroviruses able to integrate into the chromosome of non-dividing cells. Gene transfer vectors based on HIV-1 have been shown to transduce non-dividing cells effectively in vitro⁴⁶.

Several research groups have described HIV-based vectors, unfortunately the virus titers are extremely low^47,48. Recently, a much higher production of vector stock was achieved when an amphotropic envelope protein was substituted for the endogenous HIV envelope protein in trans⁴⁶. The amphotropic envelope protein, derived from either murine leukemia virus (MLV) or vesticular stomatitis virus G (VSV-G), broadened the tropism of the vector.

The use of the VSV-G envelope protein generates several problems. There is the possible production of pseudovirions where other RNAs can be encapsidated instead of recombinant virus⁴⁹. The virus titer can be over-estimated as recombinant vectors, empty vectors and pseudovirions can all be neutralized by anti-VSV antiserum.

There is no stable packaging cell line for VSV-G envelope proteins as these proteins are cytotoxic⁵⁰. The use of the MLV envelope protein generates no such problems. However, in the reported experiment, the virus titer was fourfold lower than when the VSV-G envelope was used.

Another hurdle to overcome in developing a novel recombinant lentiviral vector is the current unavailability of a stable packaging cell line. Constitutive expression of lentiviral proteins has been reported to be cytotoxic⁵¹. The use of inducible packaging cells or a cell line with a high tolerance limit may overcome this problem.

References

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