10
the nuclease can recombine during the reverse transcription of the viral genome [ 72 ].
However, using a similar approach as the zinc-finger nucleases, Cai et al. fused
TALEN proteins to viral proteins to allow incorporation into a single vector [ 69 ].
Additionally, IDLVs have been modified further to inactivate the reverse transcrip-
tase, allowing for vectors that can deliver TALEN mRNA into host genomes, avoid-
ing the possibility of recombination by viral machinery [ 73 ]. CRISPR-Cas systems
have also been delivered by IDLVs and integrating lentiviral vectors for a variety of
experiments, ranging from library-on-library screening of CRISPR-Cas cleavage
efficiency across the genome to removal of proviral DNA such as HIV-1 and Hepatitis
B [ 68 , 74 – 77 ]. In addition to nuclease delivery, an interesting application of IDLVs
has been used to deliver nucleic acid modifying enzymes such as deaminases and
epigenetic modifying enzymes such as histone deacetylases [ 56 , 78 ]. Such in situ
histone and DNA modification allows for genotypic or epigenetic change without the
introduction of foreign DNA.
1.3.3 Translational and Clinical Progress Using Retroviral
Vectors
Vectors based on retroviruses made an impact on treating human disease, and their
use may increase as vectors become increasingly safe. A landmark study exempli-
fied the success of a self-inactivating (SIN) γ-retrovirus vector to treat X-SCID [ 79 ].
This vector was an improvement on the previous generation Moloney murine leuke-
mia virus vectors used to treat X-SCID and was shown to be less mutagenic due to
a long terminal repeat (LTR) U3 enhancer deletion and the human elongation factor
1-α short promoter used to control the delivered gene [ 79 – 81 ]. Using this vector, the
group showed that 8/9 treated patients exhibited improved immune function, with
one patient dying due to a preexisting infection caused by an adenovirus [ 79 ]. Other
recent trials have shown success in the use of integrating lentiviral vectors to reduce
the autoimmune complications and microthrombocytopenia associated with
Wiskott-Aldrich syndrome [ 82 , 83 ]. One of the most promising uses of retroviral
vectors is the modification of patient T-cells to target malignant cell populations by
employing antigen receptors that bind to antigens specific to cancer cells. This
method, called chimeric antigen receptor T-cell therapy, or CAR T-cell therapy, will
be described in detail later in this chapter [ 84 ].
1.4 Adenoviral Vectors
Adenoviruses are non-enveloped double-stranded DNA viruses with ~35 kilobase
pair genomes and are somewhat larger than many viruses commonly used in gene
therapy [ 85 ]. In the adenoviral genome, several genes exist to regulate expression of
J.E. DiCarlo et al.