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identified genes to cure a disease, knocking down or deactivating problematic genes, and replacing
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malfunctioning genes with therapeutic ones . In order to transport foreign genetic material into the host
organ, gene therapy employs RNA and DNA by using different transferring vehicles/vectors. In in vivo gene
therapy, genetic material is delivered directly into target organs, while ex vivo gene therapy involves
modifying host cells outside the body before reintroducing them. The goals of gene therapy are to restrict
the activity of the affected gene, enhance the presence of modifier disease genes, or supply a functioning
gene copy of the destroyed gene [277,278] [Figure 5].

Gene editing techniques
Gene editing is a technology that makes it possible to change an organism's or cell's DNA sequence. Gene
editing can be used to improve features, develop medicines, create animal models, and rectify genetic flaws,
among other things. Nucleases, which are enzymes that can cut DNA at certain places, are essential to the
process of gene editing. Based on their structural differences, there are currently four types of nucleases that
edit genes in prostate cancer: base editors, zinc-finger nucleases (ZFN), transcription activator-like effector
nucleases (TALENs), and CRISPR-associated nucleases (CRISPR /Cas-9) [Figure 5].

Base editors
Base editors are a subset of nucleases used in gene editing that can transform one base into another without
inducing double-strand breaks (DSBs) in the DNA. Base editors consist of an adenine-to-inosine or
cytosine-to-uracil deaminase enzyme coupled with a Cas9 variant. The Cas9 variant can only bind to or
nick DNA depending on whether it is catalytically inactive (dCas9) or nickase (nCas9). The guide RNA
binding site is surrounded by a small window where the deaminase enzyme can work on the target base.
The cellular DNA repair machinery then corrects the resultant base alterations. Base editors are useful for
introducing precise base alterations or correcting point mutations in genes linked to prostate cancer, such as
androgen receptors (AR) [279,280] .

ZINC finger nucleases
Zinc finger nucleases (ZFNs) are synthetic nucleases made up of a pair of domains: a FokI nuclease domain
that breaks the DNA and a zinc finger domain that detects and binds to a particular DNA sequence. By
manipulating the zinc finger domain, which has three to six zinc finger repeats that individually recognize
three to eighteen base pairs, ZFNs can be engineered to target desired DNA sequence effectively [269,281] .
Double-strand breaks (DSBs) in DNA can be caused by ZFNs and corrected by non-homologous end
joining (NHEJ) or homology-directed repair (HDR), which involve either insertion and disruption of gene.
Plasmid-based techniques circumvent the drawbacks of viral administration, including immunogenicity,
toxicity, and insertional mutagenesis, and can transfer ZFNs to the intended cells. Certain disadvantages of
ZFNs include the potential for off-target breaks, which can result in the destruction of cells, and the random
integration of DNA donors. Additionally, when ZFNs fail to precisely target or focus on a specific spot, the
likelihood of off-target break increases. ZFNs have been employed in the modification of prostate cancer-
related genes such as AR, PTEN, and TMPRSS2-ERG [282,283] .

CRISPR-associated nucleases (CRISPR/Cas9)
Bacteria possess an adaptive immune system called CRISPR, which is heritable. This system helps the
bacteria guard against re-infection by remembering past viral infections. In contrast to the immune system
of human beings, CRISPR is inherited by the subsequent types or growth stages of bacteria, protecting the
colony from further viral infections. The functionality of the CRISPR-linked immune system requires the
[284]
binding of invader DNA (virus, plasmid) with the bacterial genome . CRISPR/Cas9 comprises Cas9
endonuclease and short noncoding guide RNA (gRNA) consisting of two parts: helper trans-activating
RNA (tracrRNA) and CRISPR RNA (crRNA) that targets specific sequences. Through base pairing between