International Journal of

Molecular Sciences

Review
Eternal Youth: A Comprehensive Exploration of Gene, Cellular,
and Pharmacological Anti-Aging Strategies
Kristina V. Kitaeva 1 , Valeriya V. Solovyeva 1 , Nataliya L. Blatt 1 and Albert A. Rizvanov 1,2, *

1 Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia;
[email protected] (K.V.K.); [email protected] (V.V.S.); [email protected] (N.L.B.)
2 Division of Medical and Biological Sciences, Tatarstan Academy of Sciences, 420111 Kazan, Russia
* Correspondence: [email protected]

Abstract: The improvement of human living conditions has led to an increase in average life ex-
pectancy, creating a new social and medical problem—aging, which diminishes the overall quality
of human life. The aging process of the body begins with the activation of effector signaling path-
ways of aging in cells, resulting in the loss of their normal functions and deleterious effects on
the microenvironment. This, in turn, leads to chronic inflammation and similar transformations in
neighboring cells. The cumulative retention of these senescent cells over a prolonged period results in
the deterioration of tissues and organs, ultimately leading to a reduced quality of life and an elevated
risk of mortality. Among the most promising methods for addressing aging and age-related illnesses
are pharmacological, genetic, and cellular therapies. Elevating the activity of aging-suppressing
genes, employing specific groups of native and genetically modified cells, and utilizing senolytic
Citation: Kitaeva, K.V.; Solovyeva,
V.V.; Blatt, N.L.; Rizvanov, A.A.
medications may offer the potential to delay aging and age-related ailments over the long term. This
Eternal Youth: A Comprehensive review explores strategies and advancements in the field of anti-aging therapies currently under
Exploration of Gene, Cellular, and investigation, with a particular emphasis on gene therapy involving adeno-associated vectors and
Pharmacological Anti-Aging cell-based therapeutic approaches.
Strategies. Int. J. Mol. Sci. 2024, 25,
643. https://doi.org/10.3390/ Keywords: aging; senolytic; senescent cells; AAV; anti-aging therapy; cell therapy; gene therapy
ijms25010643

Academic Editors: Stefano Fais and

David Della-Morte
1. Introduction
Received: 8 November 2023
Increasing life expectancy is a global trend linked primarily to better sanitation, medi-
Revised: 21 December 2023
cal advances, improved nutrition, and safer living conditions fostered by social interaction.
Accepted: 30 December 2023
Population aging poses a significant social challenge, particularly in developed countries
Published: 4 January 2024
where the proportion of the elderly population continues to rise annually. Anti-aging
Correction Statement: This article therapy, also known as rejuvenation therapy, encompasses a spectrum of interventions de-
has been republished with a minor signed to slow, reverse, or mitigate the health effects of aging. The significance of anti-aging
change. The change does not affect therapy lies in its capacity to enhance human health, improve quality of life, and extend
the scientific content of the article and
the period of healthy aging. By targeting the fundamental mechanisms of aging, anti-aging
further details are available within the
therapy aims to postpone the onset of age-related diseases and facilitate improved tissue
backmatter of the website version of
regeneration along with the normalization of physiological functions [1].
this article.
The idea, initially proposed by Peter Medawar and later encapsulated in the 1957
hypothesis of “antagonistic pleiotropy” by American evolutionist George Williams, remains
widely accepted and serves as the primary explanation for the evolution of aging today [2].
Copyright: © 2024 by the authors.
From the standpoint of classical medicine and common logic, aging is a degenerative pro-
Licensee MDPI, Basel, Switzerland. gressive process that results in tissue dysfunction and death [3]. Signs of aging, manifesting
This article is an open access article at the cellular and molecular levels, are universal across organisms and include decreased
distributed under the terms and genome stability, telomere depletion, mitochondrial dysfunction, epigenetic noise, and
conditions of the Creative Commons stem cell depletion and dysfunction [4].
Attribution (CC BY) license (https:// Several factors are known to be involved in the aging process, activating cellular
creativecommons.org/licenses/by/ withering pathways, and genetic variations, as well as epigenetic modifications, can influ-
4.0/). ence the rate of aging [5]. Certain genes are associated with longevity, while others may

Int. J. Mol. Sci. 2024, 25, 643. https://doi.org/10.3390/ijms25010643 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2024, 25, 643 2 of 21

increase susceptibility to age-related diseases. Epigenetic changes, such as DNA methy-

lation and histone modifications, can impact gene expression patterns and contribute to
aging [6]. Aging initiates at the molecular level, manifests at the cellular level, and results
in the aging of entire organ systems. For instance, aging of the adaptive immune system
leads to the development of a chronic pro-inflammatory state characterized by sluggish
inflammation, stemming from chronic activation of the innate immune system, termed “in-
flammaging” [7]. The immune system undergoes age-related changes, resulting in impaired
immune responses and heightened susceptibility to infections, autoimmune diseases, and
age-related chronic diseases [8,9]. Elevated levels of age-related proinflammatory markers
are known to be detected in most older adults, even in the absence of risk factors and
clinical activity [10]. During cellular senescence, L1 retrotransposable elements (also known
as long interspersed nuclear element (LINE)-1) become transcriptionally derepressed and
activate type I interferon (IFN)-I secretion. High IFN-I secretion is a late aging phenotype
contributing to the maintenance of the secretory phenotype associated with aging [11].
Aging has been demonstrated to contribute to the development of several autoimmune
diseases, including ANCA-associated vasculitis [12]. In aging and Alzheimer’s disease
(AD), impaired meningeal lymphatic outflow promotes the accumulation of toxic misfolded
proteins in the central nervous system (CNS). Consequently, therapeutic neutralization of
IFNγ has been shown to alleviate age-related impairment of meningeal lymphatic func-
tion [13]. It is also known that the senescence of inflammatory tumor-associated fibroblasts
(iCAF) in the tumor microenvironment leads to increased resistance of tumor cells to
chemoradiation therapy. Interleukin 1 (IL-1) inhibition or senolytic therapy has been shown
to prevent iCAF senescence and increase the sensitivity of mice to radiation. Additionally,
lower serum levels of IL-1 receptor antagonist in patients with colorectal cancer correlate
with a poor prognosis [14]. Studies have demonstrated that intra-articular injection of an
IL-17-neutralizing antibody in a model of post-traumatic arthritis reduces joint degenera-
tion and decreases the expression of the aging marker protein cyclin-dependent kinase 1A
(CDKN-1A) inhibitor, also known as p21 [15]. Systemic inflammation, identified as one of
the main but not the only markers of aging, is found in all organ systems, resulting from
pathological changes at the cellular level [16].
Under physiological conditions, senescent cells can be eliminated by the immune sys-
tem, promoting processes such as tumor suppression, embryogenesis, differentiation, and
wound healing [17]. For instance, targeted induction of cellular senescence in malignant
neoplasms is currently under active investigation: controlled senescence followed by apop-
tosis may be a crucial strategy for eliminating uncontrolled tumor cells from tissues [18–20].
Some of the most promising approaches to rejuvenate the body involve gene and cell
therapy, combined with pharmacological intervention. These approaches aim to rejuvenate
senescent cells, eliminate non-functional senescent cells, and block signaling pathways
involved in cellular aging. This review will primarily focus on gene therapy approaches
utilizing recombinant adeno-associated virus (AAV), as well as pharmacotherapy and
cell-based interventions.

2. Cellular and Organ-Specific Aging

Cellular senescence was first reported and described as early as Leonard Hayflick
and Paul Moorhead in 1961, and it was understood as a state of irreversible growth arrest
occurring in response to telomere shortening [21]. Telomeres are known to consist of 10 to
15 base pairs of tandem hexanucleotide repeats (TTAGGG) of DNA located at the ends of
linear chromosomes [22]. They serve as protective caps on the ends of chromosomes, short-
ening with each cell division, and telomere shortening is considered a sign of aging [23,24].
When a critical shortening of telomeric repeats is reached, the cell enters a state of sustained
proliferative arrest, reaching what is termed replicative senescence. Proliferation arrest is a
protective mechanism that, in the case of shortening or loss of telomeres at the ends of chro-
mosomes, prevents unplanned DNA repair reactions leading to chromosomal instability
and, consequently, malignant degeneration of the cell [25].
Int. J. Mol. Sci. 2024, 25, 643 3 of 21

Critical telomere shortening as well as mitochondrial dysfunction and genotoxic stress

lead to double-stranded DNA breaks, which activate the p53/p21 oncosuppressor signaling
pathway. High levels of p21 inhibit the kinase activity of cyclin D/cyclin-dependent kinase
(CDK)-4/6 complexes, resulting in the inhibition of cell cycle progression, whereas low
levels of p21 act as an assembly factor of the cyclin D/CDK4/6 complex and promote its
activation leading to cell cycle progression [26]. In addition to telomere shortening, there
are other mechanisms for induction of cell aging, such as through activation of oncogenes
such as BRAF (V600E mutation) or RAS, which is called oncogene-induced senescence,
or inactivation of an oncosuppressor gene such as PTEN [27,28]. This form of cellular
senescence was discovered in 1997. It was found that transfection of oncogenic HRAS (V12
mutation) into mouse, rat, and human embryonic fibroblasts resulted in the arrest of cell
proliferative activity [28,29].
The oncosuppressor gene p16, an inhibitor of cyclin-dependent kinase 4A, is specifi-
cally activated in senescent cells and suppresses CDK4/6, thereby inducing senescence-
associated cell cycle arrest in the G0/1 phase, which depends on the dephosphorylation
of retinoblastoma Rb family proteins (Rb, p107 and p130), which are known targets of
CDK4/6 [30]. P16 is one of the markers of aging and its upregulation is confirmed in many
senescent cells and tissues [31]. It is known that deletion of p16-positive senescent cells de-
lays the onset and progression of age-related pathologies in mice in vivo and prolongs the
lifespan of prematurely and naturally aging mice [32,33]. Thus, prolonged overexpression
of any of these four critical components (p53, pRB, p16INK4A and p21WAF1/CIP1 ) is sufficient
to induce senescence [34].
In addition to cell cycle arrest, senescent cells exhibit increased β-galactosidase ac-
tivity (referred to as senescence-associated β-galactosidase, SA-β-gal is one of the widely
used markers of aged cells) at pH 6.0, activation of signaling pathways, and secretion
of various growth factors, chemokines, and cytokines [35]. The activation of senescence-
associated secretory phenotype (SASP) genes and the secretion of factors such as IL-6, IL-8,
monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and
transforming growth factor-β (TGF-β) have been demonstrated to mediate epithelial cell
proliferation and play a role in the progression and development of various carcinomas [36].
Increased IL-8 secretion by senescent fibroblasts has been shown to stimulate the invasion
and metastasis of a breast cancer cell line in an in vitro cell culture [37].
Another unique phenotype of senescent cells is the formation of senescence-associated
heterochromatin foci (SAHF), which are specialized regions of facultative heterochromatin
that reduce the expression of genes promoting proliferation [38]. Epigenetic changes,
such as DNA methylation changes, histone modifications, and the expression of non-
coding RNAs, can influence gene expression patterns without altering the underlying DNA
sequence. These epigenetic modifications can impact cellular function, aging processes,
and susceptibility to age-related diseases [39].
Acute aging, associated with a programmed cellular strategy in response to various
stressors, is believed to be involved in homeostatic biological processes. However, chronic
aging is linked to prolonged exposure to cell-damaging stressors, resulting in a nonviable
and burdensome cell cycle. This places an enormous load on the reparative and regulatory
pathways required to maintain cell integrity. Unlike acute aging, chronic aging is not a
programmed response; it is persistent and is believed to play a deleterious physiological
role, contributing to aging (senility) and age-related diseases [40] (Figure 1).
Int.
Int. J.
J. Mol.
Mol. Sci.
Sci. 2024,
2024, 25,
25, x643
FOR PEER REVIEW 4 of 22
4 of 21

Figure 1. General scheme of the cell aging pathway. Exposure to stressors triggers activation of p16
and p53–p21 signaling pathways,
pathways, which
which leads
leads to
to complete
complete cell
cell aging,
aging, which
which can be completed
completed by
cell elimination from the tissue with the help of the immune system, or long-term preservation of a
pathologically functioning cell
pathologically functioning cell and
and its
its pathological
pathological influence
influence on
on the
the microenvironment in the
microenvironment in the tissue.
tissue.

As inferred
inferred from
from thethe preceding
precedingsection,
section,the theinitiation
initiationofofaging
agingatatthethecellular
cellularlevel is
level
an
is anestablished
established concept.
concept.Furthermore,
Furthermore, therethere
exist exist
studies that have
studies that delineated
have delineatedaging clocks
aging
clocks
for for specific
specific organs. organs. For instance,
For instance, algorithms algorithms
have beenhave beenbased
devised devised basedstudies
on such on suchto
studies tothe
evaluate evaluate the physiological
physiological functions offunctions
organs such of organs such as the
as the kidneys andkidneys and lungs.
lungs. These algo-
These algorithms
rithms take into
take into account the account
interplaythe interplay
between between chronological
chronological and biologicaland agesbiological
to assess
agesfunctional
the to assess the functional
status of thesestatus
organsof these
[41,42].organs
It has[41,42]. It has been demonstrated
been demonstrated that acceleratedthat
accelerated aging is characteristic of several organ systems in conditions
aging is characteristic of several organ systems in conditions such as ischemic heart dis- such as ischemic
hearthypertensive
ease, disease, hypertensive
disorders,disorders, diabetes, osteoarthritis,
diabetes, osteoarthritis, and cancer. and cancer. Moreover,
Moreover, the biologicalthe
biological age of an organ has the capacity to predict the risk of
age of an organ has the capacity to predict the risk of mortality [43]. Another question mortality [43]. Another
question to
pertains pertains to identifying
identifying the organ-specific
the organ-specific aging-related
aging-related proteinsproteins
that act that act asfactors
as causal causal
factors
in in theprocess.
the aging aging process. This consideration
This consideration is particularly
is particularly relevantrelevant
givengiven that numerous
that numerous pro-
proteins, such as KLOTHO, UMOD, MYL7, CPLX1, CPLX2,
teins, such as KLOTHO, UMOD, MYL7, CPLX1, CPLX2, and NRXN3, exhibit genetic as- and NRXN3, exhibit genetic
associations
sociations with
with diseases
diseases specifictotorespective
specific respectiveorgans
organsororare
areconfirmed
confirmedtherapeutic
therapeutictargets.
targets.
This circumstance leads to the conjecture of a potential causal role for
This circumstance leads to the conjecture of a potential causal role for these proteins in the these proteins in
the aging process [44]. During the RNA sequencing of 17 organs
aging process [44]. During the RNA sequencing of 17 organs from C57BL/6JN mice of from C57BL/6JN mice
of varying
varying ages,
ages, it was
it was observed
observed thatgenes
that genesinvolved
involvedininthetheregulation
regulation of of the
the extracellular
extracellular
matrix, binding
matrix, binding of of unfolded
unfolded proteins,
proteins, mitochondrial
mitochondrial function,
function, asas well as inflammatory
well as inflammatory and and
immune responses, are uniformly expressed across different tissues,
immune responses, are uniformly expressed across different tissues, differing only in the differing only in the
age of onset and amplitude of expression. Notably, a high correlation
age of onset and amplitude of expression. Notably, a high correlation was demonstrated was demonstrated
for vascular
for vascular cellcell adhesion
adhesion molecule-1
molecule-1 (Vcam1)
(Vcam1) in in the
the kidneys
kidneys andand fibroblast
fibroblast growth
growth factor
factor
10 (Fgf10) in the spleen, as well as for glial fibrillary acidic protein (Gfap) and brain protein.
10 (Fgf10) in the spleen, as well as for glial fibrillary acidic protein (Gfap) and brain pro-
It is also noteworthy that the level of the plasma B-cell marker immunoglobulin J chain
tein. It is also noteworthy that the level of the plasma B-cell marker immunoglobulin J
(Igj/Jchain) exhibits a persistent increase throughout life in 11 out of 17 organs. The
chain (Igj/Jchain) exhibits a persistent increase throughout life in 11 out of 17 organs. The
circadian clock genes Bhlhe40/41, Arntl, Npas2, Per3, Ciart, and Dbp also emerge as among
circadian clock genes Bhlhe40/41, Arntl, Npas2, Per3, Ciart, and Dbp also emerge as among
the top differentially expressed genes, suggesting potential involvement in metabolic
the top differentially expressed genes, suggesting potential involvement in metabolic and
and inflammatory disorders, as well as a reduction in lifespan associated with circadian
inflammatory disorders, as well as a reduction in lifespan associated with circadian
rhythm disruption [45]. In another study, it was demonstrated that aged mice exhibited
rhythm disruption [45]. In another study, it was demonstrated that aged mice exhibited
an elevation in the plasma level of von Willebrand factor (vWF). Concurrently, mRNA
an elevation in the plasma level of von Willebrand factor (vWF). Concurrently, mRNA
levels of vWF and cellular protein were significantly increased in the brain, lungs, and
levels of vWF and cellular protein were significantly increased in the brain, lungs, and
liver, but not in the kidneys and heart, of aged mice [46]. It has been discovered that aging
liver, but not in the kidneys and heart, of aged mice [46]. It has been discovered that aging
can impact chemotaxis, as reflected by levels of MCP-1, NAP-3, and eotaxin, influencing
can impact chemotaxis,
subsequent immune cellasinfiltration
reflected by in levels of MCP-1, NAP-3,
an organ-specific manner. and eotaxin,the
Notably, influencing
liver and
subsequent
spleen exhibit immune
pronouncedcell infiltration in an
effects, while organ-specific
other manner. Notably,
organs are comparatively lessthe liver [47].
affected and
spleen exhibit pronounced effects, while other organs are comparatively less affected [47].
Int. J. Mol. Sci. 2024, 25, 643 5 of 21

It is well-established that the expression of p16 increases in aged cells. For instance, in
a murine model, predominant mRNA and protein expression of p16 were demonstrated
in hepatic endothelial cells compared to non-endothelial cells in aged mice, suggesting
a functional role specifically within the liver endothelium of aged subjects. Moreover, it
has been shown that the dynamic expression of p16 may be implicated not only in aging
processes but also in ontogenetic and physiological processes [48]. Furthermore, it has
been demonstrated that p16INK4a-positive lung fibroblasts contribute to the growth of
respiratory tract stem cell organoids, suggesting their potential role in supporting epithelial
regeneration. Interestingly, the removal of p16INK4a-positive cells or the blockade of
p16INK4a expression in mice—achieved through senolytic agents or Cre-mediated deletion
from fibroblasts—resulted in a reduction in the growth of respiratory tract stem cells
induced by naphthalene-induced tissue damage and disrupted tissue repair, leading to
enhanced fibrosis [49].
However, in the context of intestinal organoids as a model for tissue regeneration, it
has been demonstrated that senescence-associated secretory phenotype (SASP) factors, par-
ticularly the secreted N-terminal domain of Ptk7, released by aging fibroblasts, dysregulate
the activity and differentiation of stem cells, ultimately disrupting crypt formation [50]. In
model systems of oxidative-stress-induced aging in vitro and ex vivo in fibroblasts of the
colon, it has been demonstrated that aging fibroblasts secrete GDF15, which promotes the
proliferation, migration, and invasion of cells in colorectal adenoma and colorectal cancer
cell lines, as well as primary colon organoids, through the MAPK and PI3K signaling path-
ways. This observation suggests a potential similarity in the pathways involved in cancer
development and aging [51]. The organ specificity of aging is supported by epigenetic data,
indicating that age-related changes in DNA methylation are also highly tissue-specific,
with the exception of CpG sites in the ELOVL2 promoter. Age-related enhancement of
DNA methylation (gain-aDMPs) accumulates on CpG islands and their flanking regions,
which are associated with the repressive PRC2 EZH2 component. These modifications may
serve as informative markers for biological age [52]. In general, the organ specificity of
aging constitutes a complex scenario wherein various organs may respond differently to
the aging process. This underscores the significance of considering this organ specificity
in the investigation of aging mechanisms and the development of strategies to maintain
health in the elderly.

3. The Use of Senolytic Agents

Senolytic drugs are designed to eliminate senescent cells—cells that can no longer
perform their functions but persist, exerting a negative impact on surrounding healthy
cells. Such cells can contribute to the development of various age-related diseases and
the associated pathological aging of the body. Research on senolytic drugs is focused on
identifying substances capable of inducing apoptosis (programmed cell death) in senescent
cells, thereby preventing their detrimental effects on healthy cells and the body as a whole.
Over 46 potentially senolytic compounds targeting senescent cell antiapoptotic pathways
(SCAP) have been identified. These include the SRC tyrosine kinase inhibitor dasatinib,
which has been approved and widely used since 2006 with a relatively good safety profile,
as well as the natural flavonoids quercetin and fisetin (refer to Table 1) [53]. These drugs
belong to the first generation of senolytics, and their action is directed at various molecular
targets and signaling pathways, such as tyrosine kinase receptors, growth factors, ephrin
B1, SRC kinases, the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) signaling
pathway, heat shock protein-90 (HSP-90), BCL-2 family members (regulators of apoptosis),
caspases, and p53 [54].
One of the best-known senolytic drugs is dasatinib, originally developed for the
treatment of blood cancers but found to be effective in killing senescent cells. Type 2
diabetes mellitus (DM2) is recognized as an age-related disease, and insulin resistance
accelerates β-cell aging [55]. We showed that senolysis of p21high cells in human adipose
ex vivo xenografted immunodeficient mice using a dasatinib + quercetin cocktail reduces
Int. J. Mol. Sci. 2024, 25, 643 6 of 21

insulin resistance [56]. Overall, this drug combination is a popular tool for increasing the
clearance of senescent cells in tissues [57–60].
In addition to dasatinib and quercetin, other drugs, some of which are also antitumor
agents, have great potential for anti-aging therapy. For example, rapamycin (sirolimus)
and its analogs (everolimus, temsirolimus, and deforolimus) bind to the cytosolic protein
FKBP12 and thus inhibit the mammalian target of rapamycin complex 1 (mTORC1), re-
ducing the incidence of malignant neoplasms in kidney transplant patients [61]. mTOR
is a serine/threonine kinase known to play a central role in the regulation of cellular
metabolism and growth by phosphorylating various substrates in response to growth
factors, stress, nutrient availability, and other stimuli. Therefore, targeting mTOR signaling
pathways is one of the promising methods for slowing aging [62].
The addition of a FOXO4-related peptide to senescent fibroblasts of the human IMR-
90 cell line has been shown to reduce their viability by more than 10-fold compared to
non-senescent IMR-90 fibroblasts or other cell types. The mechanism of action of the
FOXO4-related peptide is based on preventing the binding of the transcription factors
FOXO4 and p53 in the cell nucleus, ultimately leading to the release of the p53 protein into
the cytosol and triggering caspase-dependent apoptosis of senescent cells [63].
HSP90 is a cytoplasmic protein that prevents proteasomal degradation of AKT and
promotes lung tumor cell survival. Thus, HSP90 inhibitors are actively being studied as
a therapeutic agent for the treatment of malignant neoplasms [64]. In addition to their
antitumor activity, drugs that inhibit HSP90, such as geldanamycin, have senolytic effects
against senescent cells [65].
In some cases, senolytic compounds targeting a single SCAP node, such as inhibitors
of the BCL-2 signaling pathway (ABT-263), N (A1331852), or A1155463, tend to induce
apoptosis in a limited range of senescent cell types; however, their administration may
cause toxic effects on the body [66].
Second-generation senolytics include agents based on lysosomal and SA-β-gal-activated
drugs, nanoparticles, sodium–potassium pump gradient-dependent apoptosis activation
(Na+ /K+ -ATPase), SASP inhibitor drugs, and induction of clearance of senescent cells
by antibody–drug conjugates, vaccination, and cell therapy based on chimeric antigen
receptor synthesizing T cells (CAR T cells), which will also be mentioned in the cell therapy
section [54].
However, for the widespread use of these drugs in clinical practice, further research is
required to find the most effective combinations and verify their safety for humans.

Table 1. List of the most common pharmacologic senolytics.

Name Mechanism of Action Model Used Effect Reference

SCID-Beige mice with xenograft of Causes reversible thrombocytopenia in
BCL-XL-dependent lung mice and inhibits small-cell lung
Selective inhibitor of BCL-XL [67]
carcinoma cells of the NCI-H146 cancer xenograft growth in vivo after
lung carcinoma cell line repeated administration
Induces apoptosis of senescent
IMR-90 cell line, primary human
A-1155463 HUVEC and IMR-90 cells, but [66]
preadipocytes, HUVECs
not preadipocytes
Gastric cancer cell lines (23132/87,
SNU216, NCI-N87, MKN1, AGS,
HGC27, SNU719). Human Has a cytostatic effect on tumor cells [68]
multiple myeloma cell lines
(MM1S, KMS12PE, KMS12BM)
Mouse model of glioblastoma
A-1331852 Selective inhibitor of BCL-XL Promoted the destruction of U251 cells [69]
multiforme, U251 and SNB-19 cells
PDX-immunodeficient NMRInu/nu
mice with xenografts of A549 (lung
Cardiac Inducers of apoptosis through In vivo inhibition of xenografted
adenocarcinoma) and [70]
glycosides the Na+ /K+ -ATPase pump tumors in mice after treatment
IMR-90 (normal human lung
cells) cells
Int. J. Mol. Sci. 2024, 25, 643 7 of 21

Table 1. Cont.

Name Mechanism of Action Model Used Effect Reference

Decreased expression of the
Reduces OPRL1 gene expression
opioid nociceptin receptor gene Human neuroglia cell line T98G [71]
associated with pain syndromes
(OPRL1)
Curcumin Inhibition of the Inhibits the MAPK signaling pathway
mitogen-activated protein kinase C57BL/6 mice and primary in the liver in old mice and p38 in old
(MAPK)/transcriptional nuclear hepatocytes isolated from the mice with diet-induced obesity. [72]
factor κB (NF-κB) livers of C57BL/6 mice Improves insulin homeostasis and
signaling pathway reduces body weight in old mice
Inhibits the cell adhesion, migration,
Suppression of SRC family DU-145 and LNCaP prostate
and invasion of prostate cancer cells at [73]
kinase inhibitors cancer cells
low nanomolar concentrations
Decreased mRNA levels of genes
Dasatinib +
encoding proinflammatory
quercetin Danio rerio fish larvae deficient in
cytokines: IL-1β, tumor necrosis Has an anti-inflammatory effect and
the serine protease inhibitor [7]
factor α (TNF-α) and chemokine reduces chronic inflammation
protein Spint1a
(C-X-C motif) ligand 8b.1
(CXCL-8b.1)
Ercc1−/∆ mice (human progeroid
Tissue-specifically reduces cellular
Blocks the signaling pathway syndrome model) and aged
Fisetin senescence in mouse adipose tissue [74]
PI3K/AKT/mTOR/p16INK4a wild-type mice, human fibroblasts
and human cells
(IMR-90)
FOXO4- Blocks the interaction of Removes (eliminates) senescent cells in
Human chondrocytes of early and
related transcription factor FOXO4 with the late passage chondrocyte [75]
late passages
peptide p53, leading to apoptosis population in vitro
Primary embryonic fibroblasts Prolongs the lifespan of mice with a
from Ercc1−/− /mesenchymal stem progeria model, delays the onset of
Geldanamycin HSP90 inhibitor [76]
cells (MSCs) from the bone marrow several age-related symptoms, and
of Ercc1−/∆ and Ercc1−/∆ mice reduces the expression of p16INK4a
Human bladder cancer cell lines
T24, 5637 (with p53 mutation), and
Inhibits cell survival and induces cell
Inhibitor of mTOR RT-4 and rat bladder cancer cell
Luteolin cycle arrest in the G2/M phase; p21 [77]
signaling pathway line BC31 (with p53 mutation)
activation in bladder cancer cells
in vitro/rats with bladder
cancer model
Navitoclax
Human skin xenograft in Causes selective elimination of
(previously BCL-2 inhibitor [78]
immunodeficient mice senescent dermal fibroblasts
ABT263)
A chemically induced aging
Has a senolytic effect; reduces levels of
E3 ubiquitin ligase inhibitor mouse model, an Alu-induced
Nutlin-3a aging markers, SASP components, and [79]
MDM2/p53 geographic retinal atrophy model,
ocular pigment deposits
and aged mice
Shows moderate selectivity in reducing
Inhibitor of extracellular
Senescent human fibroblasts of the viability of
Piperlongumin signal-regulated kinase [80]
WI-38 lineage ionizing-radiation-induced senescent
(ERK) 1/2
fibroblasts of the WI-38 lineage
Increases Nrf2 levels, which activates
autophagy, and reduces induction of
Nrf2-KO fibroblasts (nuclear factor
Inhibitor of mTOR cellular senescence in vitro. In mice,
Rapamycin Nrf2 knockout) in vivo, Nrf2-KO [81]
signaling pathway Nrf2-KO reduces the concentration of
mice in vitro
proinflammatory cytokines in serum
and adipose tissue in vivo
Primary embryonic fibroblasts Reduces the number of senescent
Resorcin HSP90 inhibitor [76]
from Ercc1−/− and Ercc1−/∆ mice mouse embryonic cells
An isogenic model of BAX
knockout in human colon Causes a cytostatic antiproliferative
Tanespimycin
HSP90 inhibitor carcinoma cell line HCT116 effect on tumor cells through inhibition [82]
(17-AAG)
in vitro and in tumor xenografts of oncogenes
in vivo.
Prolongs the lifespan of Ercc1−/∆ mice,
Alvespimycin Primary embryonic fibroblasts delays the onset of several age-related
HSP90 inhibitor [76]
(17-DMAG) from Ercc1−/− and Ercc1−/∆ mice symptoms, and reduces
p16INK4a expression
Int. J. Mol. Sci. 2024, 25, 643 8 of 21

4. Cell and Genetically Modified Cell-Based Therapy

Cell therapy is considered one of the promising approaches for correcting age-related
diseases, involving the use of the regenerative and immunomodulatory properties of cells to
restore tissues’ functions and improve overall health. However, in the context of anti-aging
cell therapy, there are nuances to consider when choosing a treatment strategy. It is known
that the regenerative capacity of tissues decreases with age. For example, studies have
shown that when using cells of bone marrow origin, young recipients exhibit statistically
better skin healing efficiency than older recipients [83]. This property is manifested at
the organ level; for instance, in adult recipients, kidneys from young donors show an
advantage in engraftment and a lower risk of rejection compared to kidneys from older
donors [84]. Moreover, it has been shown that the organs of elderly donors had a negative
effect on the recipients, accelerating the aging of their bodies [85].
It was shown that human skin xenografts from elderly donors transplanted into young
immunodeficient mice were morphologically rejuvenated within the first month after trans-
plantation. However, interestingly, during the following year, skin rejuvenation leveled off,
and the transplanted sections returned to their condition before being transplanted into
mice [86]. The data from transplantation studies are consistent and confirmed by in vitro
studies. Co-culture of epithelial progenitor cells isolated from aged mice with MSCs or with
membrane vesicles isolated from the MSCs of young mice resulted in the rejuvenation of
“old” epithelial progenitor cells [87]. It was shown that cardiosphere-derived cells (CDCs),
which are cardiac progenitor cells isolated from the hearts of neonatal rats, reproduced the
juvenile pattern of gene expression when injected into the hearts of old animals. Telomeres
in heart cells were also longer in animals after CDC transplantation [88]. Injection of
cerebrospinal fluid from young mice directly into the brain induced oligodendrocyte prolif-
eration and consolidation of long-term memory in old mice [89]. In view of these findings,
using cells isolated from young donors or placing senescent cells in a medium containing
factors characteristic of a young organism may be one tool for tissue rejuvenation.
The use of stem cells demonstrates effectiveness due to their ability to differentiate into
various cell types, regenerate, or replace damaged tissues and organs [90,91]. Autologous
MSCs are considered safe and may be effective for certain conditions [92]. For example,
the inclusion of MSCs in standard therapy for the early phase of acute severe pancreatitis
in patients of the middle-aged group (approximately 44 years old) allows purposeful
and relatively quick intervention in abnormal homeostatic processes, inhibiting toxic
phenomena, restoring immune response, and improving microcirculation [93]. Positive
results were obtained using bone marrow cells multiplied in vitro and injected into the
defect site along with biphasic calcium phosphate granules, inducing the formation of
new bone. In this case, the volume of regenerated bone was sufficient for dental implant
placement in patients aged 52–79 years, with satisfactory aesthetic and functional results
and no reported side effects (NCT02751125) [94].
Moreover, MSCs and adipose-tissue stem cells offer an effective alternative to reduce
or slow down the aging process of facial skin [95]. It was shown that natural MSC exosomes
both had neuroprotective effects in the microglia culture line BV2 in vitro and exhibited anti-
apoptotic and antioxidant effects in the brains of aging SAMP8 mice with an accelerated
aging phenotype [96]. MSCs overexpressing sirtuin (SIRT) 3 improved rat cardiac function
and increased VEGF-A levels and vascular density [97]. However, despite the versatility
and safety of MSC use, questions regarding their efficacy for certain conditions, such as
osteoarthritis, remain [98].
Another option for cell therapy is the use of stromal vascular fraction (SVF) and
platelet-rich plasma (PRP). SVF can enhance angiogenesis and neovascularization in wound
healing and urogenital and cardiovascular diseases [99]. Coronary microvascular levels
of β1 adrenergic receptors are known to decrease with age, but this decrease in expres-
sion was restored by treatment with SVF. Intravenous administration of SVF to aged rats
improved their coronary microvascular function [100]. The application of autologous
microfragmented adipose tissue with SVF in patients with knee osteoarthritis increased
Int. J. Mol. Sci. 2024, 25, 643 9 of 21

glycosaminoglycan levels in hyaline cartilage in older patients (63+ years of age), resulting
in decreased pain and improved motor ability [101].
Platelet-rich plasma (PRP) is plasma with a platelet content several times higher than
that of blood plasma. It has been demonstrated that PRP stimulates the activity of glycolytic
enzymes in fibroblasts, decreases the rate of oxygen consumption, and impacts certain
types of mitochondrial respiration. Moreover, PRP activates SIRT1 expression, contributing
to the rejuvenation of fibroblasts [102]. According to clinical data, intra-articular injection
of PRP into patients with osteoarthritis did not result in adverse effects. However, due
to the peculiarities of PRP preparation and significant variations in the ratio of cellular
fractions in the final product, further studies and validation of this therapeutic method are
required [103]. Another cell variant studied for its therapeutic potential is RCS-01. This
preparation consists of 25 × 106 cultured autologous cells derived from the nebulbar dermal
sheath of a hair follicle in the anagenic state. RCS-01 injections were well tolerated, with no
reported serious side effects. A single injection of RCS-01 led to a significant increase in
the mRNA expression of TGF-β1, connective tissue growth factor (CTGF), type I collagen
(COL1) A1, COL1A2, COL3A1, and lumican genes (NCT02391935) [104].
A major trend in cell therapy involves the use of reprogrammed and genetically
modified cells. Cell reprogramming entails resetting the epigenetic state of cells, reverting
them to a younger state, with the goal of reversing cellular aging and restoring function.
For instance, the use of induced pluripotent stem cells (iPSCs) represents a promising
therapy for age-related macular degeneration, one of the leading causes of irreversible
visual impairment globally, characterized by degeneration of the retinal pigment epithelium.
Several studies have proposed therapeutic options based on retinal pigment epithelial cells
differentiated from allogeneic iPSCs, with successful trials conducted on various animal
models, including allografts in macaque monkeys [105–108].
However, not all experiments with the introduction of iPSCs have been equally suc-
cessful. Old rats injected with human neural progenitor cells derived from iPSCs at the
site of chronic cervical spinal cord injury showed no improvement in behavioral tests.
Additionally, high mortality rates were observed during behavioral training (41.2%), after
injury (63.2%), and after cell injection (50%). Meanwhile, histological analysis revealed that
the injected cells survived and remained at the transplant site, without inducing tumors,
confirming their safety [109]. Modified tendon fibroblasts expressing angiogenic factors
(placental growth factor (PlGF) and matrix metalloproteinase-9 (MMP9)) can repair the
vasculature and reduce collagen deposition, enabling effective cell therapy in aged mice
with dystrophy [110]. Genetically modified effector immune cells, such as CAR T cells tar-
geting the urokinase-type plasminogen activator surface receptor (uPAR), induced during
senescence, have shown high efficacy in removing senescent cells both in vitro and in vivo,
as well as in restoring tissue homeostasis in mice with induced fibrosis [111]. Thus, one of
the key aspects of implementing cell therapy lies in its potential role in the prevention of
age-related diseases, such as cancer, heart diseases, and immune system dysfunction. Tissue
regeneration and the maintenance of tissue health can play a crucial role in reducing the
risk of developing these conditions. It is noteworthy that despite the promising prospects
of cell therapy, this approach is still in the active research stage. Further investigations
and developments are required for a comprehensive understanding of the capabilities and
limitations of cell therapy in the context of combating aging. The safety and effectiveness
of this approach remain pivotal aspects demanding careful scrutiny.

5. Possibilities for Gene Therapy Based on Recombinant Adeno-Associated Viruses

Recombinant AAVs are small, non-enveloped viruses, with the deletion of the rep and
cap genes replaced by the insertion of the transgene of interest. The viral vectors contain
4.7 kb single-stranded genomic DNA with two palindromic GC-rich inverted end repeats
at the ends of the chain [112]. Recombinant viruses are constructs comprising a promoter,
genes of interest, and a terminator to enhance their suitability for clinical applications. As
these recombinant AAVs cannot replicate, they provide a safe means for ensuring long-
Int. J. Mol. Sci. 2024, 25, 643 10 of 21

term transgene expression following a single infection. AAVs have emerged as efficient
carriers for genetic modification due to their effective in vivo infectivity, non-pathogenic
nature, broad tissue tropism, infrequent genomic integration, and ability to infect and
persist in non-dividing cells [113,114]. Four decades of research have demonstrated that
AAVs are among the safest and most effective vectors for delivering genes of interest to a
diverse array of cell types in gene therapy applications [115,116]. Hereditary diseases are
particularly attractive targets, and AAV vectors are well-established as prominent genetic
therapies, including therapies for aging [113,117].
Several genes have been identified as potential targets for gene therapy aimed at
enhancing longevity and health. These genes are often involved in signaling pathways that
play a role in the regulation of cellular metabolism, oxidative stress, and inflammation, all of
which are believed to contribute to the aging process. In this review, we will explore some of
these genes, along with the possibilities of utilizing AAVs to address age-related diseases.

5.1. Sirtuins
Sirtuins constitute a family of proteins that play a crucial role in the regulation of
cellular metabolism and stress response. Notably, Sirt1 overexpression, particularly in
skeletal muscle, has been demonstrated to counteract the development of insulin resistance
induced by a high-fat diet in mice. While the administration of AAV1-Sirt1 alone did not
suffice to prevent obesity and insulin resistance caused by a high-fat diet, it was observed
that there was an increased expression of key genes related to β-oxidation, accompanied
by elevated levels of phosphorylated AMP-activated protein kinase (AMPK). Furthermore,
Sirt1 overexpression in skeletal muscle led to an augmentation of basal levels of AKT
phosphorylation [118]. Additionally, intravitreal delivery of Sirt1 improved visual function
in mice with diabetic retinopathy [119].
SIRT3 is broadly expressed in mitochondria-rich tissues with high metabolic demands,
including brain, heart, kidney, muscle, and brown adipose tissue. Particularly in cells such
as neurons, cardiomyocytes, and hepatocytes, SIRT3 plays a protective role against inflam-
mation, oxidative stress, and senescence. For instance, gene expression and phenotype
analysis of Sirt3 knockout mice revealed that reducing Sirt3 expression mediated persistent
inflammation in the liver. Conversely, restoring Sirt3 expression in the liver effectively inhib-
ited persistent liver inflammation and cardiovascular damage [120]. It was demonstrated
that SIRT3 expression was suppressed in senescent human MSCs. CRISPR/Cas9-mediated
inhibition of SIRT3 resulted in impaired nuclear integrity, loss of heterochromatin, and the
accelerated aging of MSCs. SIRT3 was also shown to interact with nuclear envelope proteins
and proteins associated with heterochromatin. SIRT3 deficiency led to the separation of
laminin-associated genomic domains from the nuclear lamina, increased chromatin accessi-
bility, and aberrant transcription of repetitive sequences [121]. In another study, receptor
activator of NF-κB ligand (RANKL) was demonstrated to increase Sirt3 mRNA and protein
levels, thereby stimulating osteoclast formation and bone resorption, most likely through
the stimulation of mitochondrial metabolism. In the absence of Sirt3, the mitochondrial
function of osteoclasts decreases with age, resulting in reduced osteoclast function and the
preservation of bone mass in mice [122]. Suppression of SIRT3 was found to further increase
lactate dehydrogenase release, decrease ATP levels, suppress mitochondrial membrane
potential, and increase oxidative stress in cardiomyocytes. SIRT3 deficiency further ele-
vated the expression of necroptosis-related proteins, including receptor-interacting protein
kinase 1 (RIPK-1), RIPK3, and cleaved caspase 3 (CASP-3), and increased the expression of
inflammation-related genes, including NOD-like receptor family pyrin-domain-containing
protein 3 (NLRP3), CASP1, p20, and IL-1β both in vitro and in vivo [123]. It was demon-
strated that topical irisin treatment reduced alveolar bone loss and oxidative stress while
increasing Sirt3 expression in periodontal tissues in experimentally induced rat models
of diabetes and periodontitis. By culturing periodontal ligament cells (PDLCs) in vitro,
it was found that irisin could partially restore suppressed cell viability, reduce intracellu-
lar oxidative stress and mitochondrial dysfunction, and restore impaired osteogenic and
Int. J. Mol. Sci. 2024, 25, 643 11 of 21

osteoclastogenic abilities in PDLCs upon exposure to high glucose and proinflammatory

stimulation [124]. However, caution should be exercised in the use of a strategy to increase
SIRT3 expression, ensuring tissue specificity to avoid exacerbating pathological effects.
SIRT6 overexpression has been shown to preserve telomere integrity, delay cellular
senescence, and reduce the expression of senescence-associated inflammatory cytokines
and changes in vascular smooth muscle cell metabolism. SIRT6 promoted the proliferation
and longevity of murine vascular smooth muscle cells and prevented metabolic changes
associated with aging. However, the mutant variant SIRT6H133Y (inactive deacetylase
mutant variant of SIRT6) shortened the lifespan of both human and murine vascular smooth
muscle cells [125]. Nevertheless, overexpression of SIRT6 has been demonstrated to induce
carotid plaque hemorrhage by promoting angiogenesis in atherosclerotic plaques through
increased expression of hypoxia-inducible factor 1α (HIF-1α) under hypoxic conditions,
in conjunction with damage to newly formed vessels via reactive oxygen species under
oxidative stress [126]. Thus, SIRT1 appears to be the most promising and safest target
within the sirtuin family at this time.

5.2. Telomerase
Telomerase is an enzyme that plays a crucial role in maintaining the length of telomeres,
protective caps at the ends of chromosomes. Telomere shortening is believed to contribute to
the aging process. Researchers have explored the use of viral vectors to deliver telomerase-
expressing genes into cells with the aim of slowing telomere shortening and promoting
longevity. While there is considerable variation in telomere length between individuals,
telomeres inevitably shorten with age and cell division [127]. A method for enhancing
longevity using a cytomegalovirus vector encoding telomerase reverse transcriptase (TERT)
and follistatin (FST) genes has been proposed and demonstrated to be highly effective in
a mouse model of natural aging. When administered intranasally or by injection, gene
therapy resulted in increased longevity (by more than 32%), improved glucose tolerance
and physical endurance, and prevented weight loss and alopecia areata [128].
The application of gene therapy to express active human TERT (hTERT) in human cells
holds the potential to treat numerous neurodegenerative diseases associated with aging,
including Alzheimer’s disease (AD). Clinical trials involving this strategy are underway,
such as one conducted by Libella Gene Therapeutics. This trial involves treatment with
hTERT delivered via transduction using adeno-associated virus (AAV) (NCT04133454).
The objective is to lengthen telomeres to prevent, delay, or even reverse the progression
of AD. Telomere lengthening is anticipated to have a direct impact on cognitive function
and the quality of life in patients with age-related neurodegenerative diseases such as AD.
However, the use of hTERT is accompanied by the risk of malignant cell transformation. For
instance, the hTERT/MDM2-FOXO3a-integrin β1 (ITGB1) signaling pathway is implicated
in hTERT-stimulated gastric cancer invasion, suggesting that this signaling pathway may
be a novel target for the prevention and treatment of gastric cancer metastasis [129,130].

5.3. The Aging Suppressor Gene Klotho

Klotho is an aging suppressor gene whose association with longevity has been demon-
strated in animal models. It is known that the amount of Klotho decreases with age
in humans and mice, while increasing its expression slows down or reverses age-related
changes [131]. The Klotho gene encodes a type I membrane protein related to β-glucosidases.
Reduced production of this protein has been observed in patients with chronic renal failure
(CRF), and this may be one of the factors underlying the degenerative processes (e.g., arte-
riosclerosis, osteoporosis, and skin atrophy) observed in CRF. Additionally, mutations in
this protein are associated with aging and loss of bone mass (GeneID: 9365). The effect of a
deficiency of this protein was demonstrated in a mouse model with Klotho haplodeficiency,
which caused impaired kidney function [132]. Klotho deficiency leads to heart failure in
Klotho-hypomorphic mutant (KL(−/−) ) mice [133]. In this study, we demonstrated that
Int. J. Mol. Sci. 2024, 25, 643 12 of 21

intra-articular delivery of the α-Klotho gene using plasmid DNA increased Klotho protein
synthesis and delayed cartilage degradation in a mouse model of osteoarthritis [134,135].
The use of gene therapy to enhance Klotho activity in humans using AAV is actively
being explored. For example, AAV-Klotho was injected into the bilateral hippocampus of
rats with a model of temporal lobe epilepsy, and after 9 weeks, it was found that AAV-
Klotho induced Klotho overexpression in the hippocampus, effectively improved cognitive
impairment, and had a neuroprotective effect. Additionally, Klotho significantly increased
glutathione peroxidase-4 and glutathione levels while suppressing reactive oxygen species
levels in a rat model of temporal lobe epilepsy [136]. It was shown that the administration
of AAV-Klotho to mice with a temporal lobe epilepsy model significantly attenuated hip-
pocampal neuronal damage and cognitive impairment [137]. AAV-mKlotho (murine Klotho)
prevented the progression of spontaneous hypertension, eliminated renal tubule atrophy
and dilatation, and attenuated kidney damage in rats with spontaneous hypertension [138].
Neuroprotective and anti-inflammatory effects, as well as the restoration of the epigenetic
landscape upon the administration of AAV9-Klotho to mice with a rapid aging model, were
confirmed [139].
The administration of AAVs encoding a soluble form of the Klotho protein reduced
arterial stiffness in aging mice, including by restoring the B-cell population and serum
immunoglobulin G (IgG) levels, and attenuated aging-related vascular inflammation and
arterial remodeling [140]. There is a known trial conducted on patients with mild to
moderate dementia using AAV vectors encoding hTERT and KLOTHO, where the safety of
the vectors and improvement of cognitive function were shown; however, official data on
this study are not yet available [141]. Despite encouraging results after correcting Klotho
protein levels using AAV vectors, it has not yet been possible to overcome the molecular
entropy that inevitably occurs during the life of a cell and organism as a whole [142].

5.4. Fibroblast Growth Factor 21

Fibroblast growth factor 21 (FGF21) is a hormone that plays a crucial role in the
regulation of glucose and lipid metabolism. Studies in animal models have demonstrated
that increasing FGF21 activity can enhance metabolic function and promote longevity.
Ongoing investigations into gene therapy to elevate FGF21 activity in humans consider it
a promising therapeutic approach for the treatment of type 2 diabetes (DM2) and obesity.
Gene therapy involving AAV-FGF21 has resulted in significant reductions in body weight,
adipose tissue hypertrophy, inflammation, hepatic steatosis, inflammation, fibrosis, and
insulin resistance for over one year in animal models subjected to a high-fat diet or ob/ob
mice (obese mice) over extended periods. This therapeutic effect was achieved without side
effects, despite maintaining elevated serum FGF21 levels persistently [143]. Additionally,
mice overexpressing FGF21 exhibited a reduction in age-related thymus lipoatrophy [144].
However, there is evidence suggesting that circulating FGF21 levels increase with age
in rodents and humans. The positive metabolic effects of FGF21 administration, known
for promoting health, coincide with elevated hormone levels observed in obesity and
diabetes. This increase may be attributed to altered tissue sensitivity to FGF21 [145].
Positive outcomes were observed in a gene therapy trial utilizing three different AAVs
encoding FGF21, αKlotho, and the soluble form of mouse TGF-β receptor 2 (sTGFβR2)
genes. The trial evaluated their effectiveness in mitigating the effects of four age-related
diseases: obesity, DM2, heart failure, and kidney failure in animal models. Results included
a 58% increase in cardiac function in mice with heart failure induced by ascending aortic
constriction, a 38% decrease in α-smooth muscle actin expression, a 75% reduction in
renal medullary atrophy in mice subjected to unilateral ureteral obstruction, and complete
reversal of obesity and diabetes phenotypes in mice on a continuous high-fat diet [146].
Int. J. Mol. Sci. 2024, 25, 643 13 of 21

5.5. Using Other Genes to Treat Age-Related Diseases

5.5.1. Treatment of Age-Related Macular Degeneration
VEGF-A-mediated signaling is recognized as necessary and sufficient for the rejuve-
nation of a rapidly aging human organ, such as the skin, both at the morphological and
molecular aging marker levels [86]. Furthermore, in mice treated with AAV-VEGF, mito-
chondrial dysfunction, metabolic disorders, endothelial cell senescence, and inflammation
were reduced. This treatment resulted in increased longevity, decreased abdominal fat
accumulation, reduced liver steatosis, mitigation of muscle mass loss (sarcopenia), bone
mass loss (osteoporosis), decreased kyphosis, and a lower incidence of spontaneous tumors.
These findings suggest that VEGF could be a promising target for anti-aging therapy [147].
However, certain age-related diseases require VEGF blocking, such as age-related macular
degeneration of the yellow spot (AMD), a leading cause of blindness in the elderly. The
gene therapy product ADVM-022 (AAV.7m8-aflibercept) is being developed for AMD
treatment. A single intravenous injection of ADVM-022 has the potential to treat the wet
form of AMD by providing sustained expression of therapeutic levels of intraocular anti-
VEGF protein (aflibercept) and maintaining patients’ vision. ADVM-022 aims to reduce the
ongoing treatment burden that often leads to treatment failure and vision loss in patients
with wet AMD receiving anti-VEGF therapy in clinical practice (NCT03748784). Another
clinical trial is underway in a similar direction to block VEGF signaling in AMD patients
(NCT05657301).
A gene therapy option has been proposed using AAV1-BACE1 (encoding β-secretase)
for injection into the pigment epithelium. In mice with a retinal macular degeneration phe-
notype (superoxide dismutase 2 (SOD2) knockdown mice), AAV1-BACE1 administration
prevented the loss of retinal function and pathology, maintaining these effects for up to 6
months. Additionally, BACE1 overexpression inhibited oxidative stress, microglia changes,
and loss of the integrity of tight contacts in the retinal pigment epithelium in mice [148].
Therapy with AAV-APNp1 (encoding adiponectin peptide 1) was also evaluated in mice
with a model of AMD. The results showed increased APNp1 expression in the retina and
vasculature over a 28-day period, which could be considered for further clinical trials [149].
Anti-inflammatory gene constructs, such as AAV-TatCARD, may also be considered for
the treatment of inflammation in AMD and other posterior pole ocular diseases where
inflammation may play a role [150]. The efficacy of gene therapy for AMD using subretinal
delivery of AAV-NDI1-7 has been demonstrated. Additionally, this gene has shown utility
in studies involving Parkinson’s disease, Leber’s hereditary optic neuropathy, and multiple
sclerosis, reducing oxidative stress in these conditions [151].

5.5.2. Cognitive Dysfunction/Neurodegenerative Diseases

A clinical trial (Phase 1) of gene therapy utilizing AAV2-BDNF (encoding brain-
derived neurotrophic factor) to address early Alzheimer’s disease (AD) and mild cognitive
impairment in 12 participants is currently ongoing. BDNF, a nervous system growth factor,
regulates neuronal function in key memory circuits of the brain (entorhinal cortex and
hippocampus), potentially enhancing cognitive function in patients [152] (NCT05040217).
Astroglial expression of clusterin (Clu) mediated by AAV was found to promote excitatory
neurotransmission in wild-type mice and restore synaptic deficits in Clu knockout mice.
Overexpression of Clu in astrocytes of 5xFAD mice with a BA model resulted in reduced
β-amyloid accumulation and complete restoration of synaptic deficits [153].
CERE-120, a gene product (AAV2 encoding the neurotrophic factor neurturin), has
been developed to deliver a therapeutic factor to degenerating nigrostriatal neurons in
Parkinson’s disease. While it has been shown to be safe, ongoing investigation in phase
2 clinical trials is examining its efficacy [154]. Additionally, PBFT02, an AAV1-based
drug, is currently in clinical trials for gene therapy targeting frontal temporal dementia
by delivering a functional copy of the human progranulin (GRN) gene into the brain. The
study aims to assess the safety, tolerability, and efficacy of this treatment in patients with
frontal temporal dementia and mutations in the GRN gene (NCT04747431).
Int. J. Mol. Sci. 2024, 25, 643 14 of 21

Gene therapy has shown promise in improving the condition of animals with peri-
operative neurocognitive disorder. Downregulation of myeloid differentiation factor 2
(MD2) expression by AAV-shMD2 (encoding hairpin RNA) or injection of the synthetic
MD2-disrupting peptide Tat-CIRP-CMA improved spatial reference learning ability and
memory in anesthetized and surgically treated animals [155]. Finally, intracerebroventricu-
lar infusion of nerve growth factor (NGF) into the basal forebrain improved spatial memory
in old animals. Alongside preventing age-related memory deficits, NGF gene transfer
increased the size of cholinergic neurons by 34% in the medial septum. This approach may
represent an effective therapy for age-related dementias associated with the dysfunction of
cholinergic activity and memory, such as AD [156].

6. Conclusions
The increase in life expectancy is a positive trend in human history, but aging has
become a prominent challenge that we must address using new technologies and precise
tools. The emerging focus on senolytics, substances capable of removing senescent cells
from the body, stems from successful clinical trials of some drugs. Senescent cells play a
pivotal role in age-related diseases, and their elimination can offer significant therapeutic
benefits. However, most senolytics currently exhibit serious side effects that need to be
addressed for optimal results.
Cell therapy, involving the use of stem cells and specialized cells to repair damaged
and aging tissues, holds promise for slowing the aging process and enhancing overall
health. However, when using autologous stem cells from elderly patients, obtaining an
unspecified therapeutic effect is necessary due to their intrinsic senescent nature.
The use of AAV to deliver genetic material and a gene that can slow down the aging
process may be a revolutionary approach to treating aging, opening new horizons for
slowing down and even reversing its processes. However, this is contingent on eliminating
undesirable side effects and reducing production costs. While the studies described above
have demonstrated inspiring success in improving the physical condition of model animals
and patients, the centuries-long quest for a true “elixir of youth” to reverse age-related
changes and eliminate diseases is far from over.

Author Contributions: Conceptualization, K.V.K. and V.V.S.; writing—review and editing, N.L.B.
and K.V.K.; supervision, V.V.S. and A.A.R. All authors have read and agreed to the published version
of the manuscript.
Funding: This study was funded by the subsidy allocated to Kazan Federal University for state
assignment №FZSM-2023-0011 in the sphere of scientific activities.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data available in a publicly accessible repository.
Acknowledgments: This work is part of the Kazan Federal University Strategic Academic Leadership
Program (PRIORITY-2030).
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Ros, M.; Carrascosa, J.M. Current nutritional and pharmacological anti-aging interventions. Biochim. Biophys. Acta Mol. Basis Dis.
2020, 1866, 165612. [CrossRef]
2. Austad, S.N.; Hoffman, J.M. Is antagonistic pleiotropy ubiquitous in aging biology? Evol. Med. Public Health 2018, 2018, 287–294.
[CrossRef] [PubMed]
3. Hernandez-Segura, A.; Nehme, J.; Demaria, M. Hallmarks of Cellular Senescence. Trends Cell Biol. 2018, 28, 436–453. [CrossRef]
[PubMed]
4. Ocampo, A.; Reddy, P.; Belmonte, J.C.I. Anti-Aging Strategies Based on Cellular Reprogramming. Trends Mol. Med. 2016, 22,
725–738. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2024, 25, 643 15 of 21

5. Pagliai, G.; Sofi, F.; Dinu, M.; Sticchi, E.; Vannetti, F.; Molino Lova, R.; Ordovas, J.M.; Gori, A.M.; Marcucci, R.; Giusti, B.; et al.
CLOCK gene polymorphisms and quality of aging in a cohort of nonagenarians—The MUGELLO Study. Sci. Rep. 2019, 9, 1472.
[CrossRef] [PubMed]
6. Crouch, J.; Shvedova, M.; Thanapaul, R.; Botchkarev, V.; Roh, D. Epigenetic Regulation of Cellular Senescence. Cells 2022, 11, 672.
[CrossRef] [PubMed]
7. Hernandez-Silva, D.; Canton-Sandoval, J.; Martinez-Navarro, F.J.; Perez-Sanchez, H.; de Oliveira, S.; Mulero, V.; Alcaraz-Perez, F.;
Cayuela, M.L. Senescence-Independent Anti-Inflammatory Activity of the Senolytic Drugs Dasatinib, Navitoclax, and Venetoclax
in Zebrafish Models of Chronic Inflammation. Int. J. Mol. Sci. 2022, 23, 10468. [CrossRef] [PubMed]
8. Franceschi, C.; Garagnani, P.; Parini, P.; Giuliani, C.; Santoro, A. Inflammaging: A new immune-metabolic viewpoint for
age-related diseases. Nat. Rev. Endocrinol. 2018, 14, 576–590. [CrossRef]
9. Noguchi, M.; Kodama, T.; Shimosato, Y.; Koide, T.; Naruke, T.; Singh, G.; Katyal, S.L. Papillary adenoma of type 2 pneumocytes.
Am. J. Surg. Pathol. 1986, 10, 134–139. [CrossRef]
10. Michaud, M.; Balardy, L.; Moulis, G.; Gaudin, C.; Peyrot, C.; Vellas, B.; Cesari, M.; Nourhashemi, F. Proinflammatory cytokines,
aging, and age-related diseases. J. Am. Med. Dir. Assoc. 2013, 14, 877–882. [CrossRef]
11. De Cecco, M.; Ito, T.; Petrashen, A.P.; Elias, A.E.; Skvir, N.J.; Criscione, S.W.; Caligiana, A.; Brocculi, G.; Adney, E.M.; Boeke, J.D.;
et al. L1 drives IFN in senescent cells and promotes age-associated inflammation. Nature 2019, 566, 73–78. [CrossRef] [PubMed]
12. Alikhan, M.A.; Jaw, J.; Shochet, L.R.; Robson, K.J.; Ooi, J.D.; Brouwer, E.; Heeringa, P.; Holdsworth, S.R.; Kitching, A.R. Ageing
enhances cellular immunity to myeloperoxidase and experimental anti-myeloperoxidase glomerulonephritis. Rheumatology 2022,
61, 2132–2143. [CrossRef]
13. Rustenhoven, J.; Pavlou, G.; Storck, S.E.; Dykstra, T.; Du, S.; Wan, Z.; Quintero, D.; Scallan, J.P.; Smirnov, I.; Kamm, R.D.; et al.
Age-related alterations in meningeal immunity drive impaired CNS lymphatic drainage. J. Exp. Med. 2023, 220, e20221929.
[CrossRef] [PubMed]
14. Nicolas, A.M.; Pesic, M.; Engel, E.; Ziegler, P.K.; Diefenhardt, M.; Kennel, K.B.; Buettner, F.; Conche, C.; Petrocelli, V.; Elwakeel,
E.; et al. Inflammatory fibroblasts mediate resistance to neoadjuvant therapy in rectal cancer. Cancer Cell 2022, 40, 168–184.e13.
[CrossRef] [PubMed]
15. Faust, H.J.; Zhang, H.; Han, J.; Wolf, M.T.; Jeon, O.H.; Sadtler, K.; Pena, A.N.; Chung, L.; Maestas, D.R., Jr.; Tam, A.J.; et al. IL-17
and immunologically induced senescence regulate response to injury in osteoarthritis. J. Clin. Investig. 2020, 130, 5493–5507.
[CrossRef]
16. Amarya, S.; Singh, K.; Sabharwal, M. Ageing Process and Physiological Changes. In Gerontology; InTech: London, UK, 2018.
17. Burton, D.G.; Krizhanovsky, V. Physiological and pathological consequences of cellular senescence. Cell Mol. Life Sci. 2014, 71,
4373–4386. [CrossRef]
18. Wang, L.; Lankhorst, L.; Bernards, R. Exploiting senescence for the treatment of cancer. Nat. Rev. Cancer 2022, 22, 340–355.
[CrossRef]
19. Park, S.S.; Choi, Y.W.; Kim, J.H.; Kim, H.S.; Park, T.J. Senescent tumor cells: An overlooked adversary in the battle against cancer.
Exp. Mol. Med. 2021, 53, 1834–1841. [CrossRef]
20. Missiaen, R.; Anderson, N.M.; Kim, L.C.; Nance, B.; Burrows, M.; Skuli, N.; Carens, M.; Riscal, R.; Steensels, A.; Li, F.; et al. GCN2
inhibition sensitizes arginine-deprived hepatocellular carcinoma cells to senolytic treatment. Cell Metab. 2022, 34, 1151–1167.e1157.
[CrossRef]
21. Hayflick, L.; Moorhead, P.S. The serial cultivation of human diploid cell strains. Exp. Cell Res. 1961, 25, 585–621. [CrossRef]
22. Saretzki, G. Telomeres, Telomerase and Ageing. Subcell. Biochem. 2018, 90, 221–308. [CrossRef] [PubMed]
23. Campisi, J.; Kim, S.H.; Lim, C.S.; Rubio, M. Cellular senescence, cancer and aging: The telomere connection. Exp. Gerontol. 2001,
36, 1619–1637. [CrossRef] [PubMed]
24. Zhu, Y.; Liu, X.; Ding, X.; Wang, F.; Geng, X. Telomere and its role in the aging pathways: Telomere shortening, cell senescence
and mitochondria dysfunction. Biogerontology 2019, 20, 1–16. [CrossRef] [PubMed]
25. Gisselsson, D.; Pettersson, L.; Hoglund, M.; Heidenblad, M.; Gorunova, L.; Wiegant, J.; Mertens, F.; Dal Cin, P.; Mitelman, F.;
Mandahl, N. Chromosomal breakage-fusion-bridge events cause genetic intratumor heterogeneity. Proc. Natl. Acad. Sci. USA
2000, 97, 5357–5362. [CrossRef] [PubMed]
26. Deng, T.; Yan, G.; Song, X.; Xie, L.; Zhou, Y.; Li, J.; Hu, X.; Li, Z.; Hu, J.; Zhang, Y.; et al. Deubiquitylation and stabilization of
p21 by USP11 is critical for cell-cycle progression and DNA damage responses. Proc. Natl. Acad. Sci. USA 2018, 115, 4678–4683.
[CrossRef] [PubMed]
27. Jung, S.H.; Hwang, H.J.; Kang, D.; Park, H.A.; Lee, H.C.; Jeong, D.; Lee, K.; Park, H.J.; Ko, Y.G.; Lee, J.S. mTOR kinase leads to
PTEN-loss-induced cellular senescence by phosphorylating p53. Oncogene 2019, 38, 1639–1650. [CrossRef] [PubMed]
28. Chandeck, C.; Mooi, W.J. Oncogene-induced cellular senescence. Adv. Anat. Pathol. 2010, 17, 42–48. [CrossRef]
29. Serrano, M.; Lin, A.W.; McCurrach, M.E.; Beach, D.; Lowe, S.W. Oncogenic ras provokes premature cell senescence associated
with accumulation of p53 and p16INK4a. Cell 1997, 88, 593–602. [CrossRef]
30. Takasugi, M.; Yoshida, Y.; Hara, E.; Ohtani, N. The role of cellular senescence and SASP in tumour microenvironment. FEBS J.
2023, 290, 1348–1361. [CrossRef]
Int. J. Mol. Sci. 2024, 25, 643 16 of 21

31. Safwan-Zaiter, H.; Wagner, N.; Wagner, K.D. P16INK4A-More Than a Senescence Marker. Life 2022, 12, 1332. [CrossRef]
32. Baker, D.J.; Wijshake, T.; Tchkonia, T.; LeBrasseur, N.K.; Childs, B.G.; van de Sluis, B.; Kirkland, J.L.; van Deursen, J.M. Clearance
of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 2011, 479, 232–236. [CrossRef] [PubMed]
33. Baker, D.J.; Childs, B.G.; Durik, M.; Wijers, M.E.; Sieben, C.J.; Zhong, J.; Saltness, R.A.; Jeganathan, K.B.; Verzosa, G.C.; Pezeshki,
A.; et al. Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature 2016, 530, 184–189. [CrossRef] [PubMed]
34. McConnell, B.B.; Starborg, M.; Brookes, S.; Peters, G. Inhibitors of cyclin-dependent kinases induce features of replicative
senescence in early passage human diploid fibroblasts. Curr. Biol. 1998, 8, 351–354. [CrossRef] [PubMed]
35. Acosta, J.C.; Banito, A.; Wuestefeld, T.; Georgilis, A.; Janich, P.; Morton, J.P.; Athineos, D.; Kang, T.W.; Lasitschka, F.; Andrulis, M.;
et al. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat. Cell Biol. 2013, 15,
978–990. [CrossRef] [PubMed]
36. Horsley, G.C. Baggage from the past. Am. J. Nurs. 1988, 88, 60–63. [PubMed]
37. Ortiz-Montero, P.; Londono-Vallejo, A.; Vernot, J.P. Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-
reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line. Cell
Commun. Signal 2017, 15, 17. [CrossRef] [PubMed]
38. Olan, I.; Handa, T.; Narita, M. Beyond SAHF: An integrative view of chromatin compartmentalization during senescence. Curr.
Opin. Cell Biol. 2023, 83, 102206. [CrossRef] [PubMed]
39. Lopez-Otin, C.; Blasco, M.A.; Partridge, L.; Serrano, M.; Kroemer, G. Hallmarks of aging: An expanding universe. Cell 2023, 186,
243–278. [CrossRef]
40. Jaul, E.; Barron, J. Age-Related Diseases and Clinical and Public Health Implications for the 85 Years Old and Over Population.
Front. Public Health 2017, 5, 335. [CrossRef]
41. Belur Nagaraj, S.; Kieneker, L.M.; Pena, M.J. Kidney Age Index (KAI): A novel age-related biomarker to estimate kidney function
in patients with diabetic kidney disease using machine learning. Comput. Methods Programs Biomed. 2021, 211, 106434. [CrossRef]
42. Morris, J.F.; Temple, W. Spirometric “lung age” estimation for motivating smoking cessation. Prev. Med. 1985, 14, 655–662.
[CrossRef] [PubMed]
43. Tian, Y.E.; Cropley, V.; Maier, A.B.; Lautenschlager, N.T.; Breakspear, M.; Zalesky, A. Heterogeneous aging across multiple organ
systems and prediction of chronic disease and mortality. Nat. Med. 2023, 29, 1221–1231. [CrossRef]
44. Oh, H.S.; Rutledge, J.; Nachun, D.; Palovics, R.; Abiose, O.; Moran-Losada, P.; Channappa, D.; Urey, D.Y.; Kim, K.; Sung, Y.J.; et al.
Organ aging signatures in the plasma proteome track health and disease. Nature 2023, 624, 164–172. [CrossRef]
45. Schaum, N.; Lehallier, B.; Hahn, O.; Palovics, R.; Hosseinzadeh, S.; Lee, S.E.; Sit, R.; Lee, D.P.; Losada, P.M.; Zardeneta, M.E.; et al.
Ageing hallmarks exhibit organ-specific temporal signatures. Nature 2020, 583, 596–602. [CrossRef] [PubMed]
46. Alavi, P.; Yousef Abdualla, R.; Brown, D.; Mojiri, A.; Nagendran, J.; Lewis, J.; Bourque, S.L.; Jahroudi, N. Aging Is Associated
With Organ-Specific Alterations in the Level and Expression Pattern of von Willebrand Factor. Arterioscler. Thromb. Vasc. Biol.
2023, 43, 2183–2196. [CrossRef]
47. Schadel, P.; Troisi, F.; Czapka, A.; Gebert, N.; Pace, S.; Ori, A.; Werz, O. Aging drives organ-specific alterations of the inflammatory
microenvironment guided by immunomodulatory mediators in mice. FASEB J. 2021, 35, e21558. [CrossRef] [PubMed]
48. Safwan-Zaiter, H.; Wagner, N.; Michiels, J.F.; Wagner, K.D. Dynamic Spatiotemporal Expression Pattern of the Senescence-
Associated Factor p16Ink4a in Development and Aging. Cells 2022, 11, 541. [CrossRef] [PubMed]
49. Strzyz, P. Senescent fibroblasts support lung repair. Nat. Rev. Mol. Cell Biol. 2022, 23, 775. [CrossRef]
50. Yun, J.; Hansen, S.; Morris, O.; Madden, D.T.; Libeu, C.P.; Kumar, A.J.; Wehrfritz, C.; Nile, A.H.; Zhang, Y.; Zhou, L.; et al.
Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling. Nat.
Commun. 2023, 14, 156. [CrossRef]
51. Guo, Y.; Ayers, J.L.; Carter, K.T.; Wang, T.; Maden, S.K.; Edmond, D.; Newcomb, P.P.; Li, C.; Ulrich, C.; Yu, M.; et al. Senescence-
associated tissue microenvironment promotes colon cancer formation through the secretory factor GDF15. Aging Cell 2019,
18, e13013. [CrossRef]
52. Slieker, R.C.; Relton, C.L.; Gaunt, T.R.; Slagboom, P.E.; Heijmans, B.T. Age-related DNA methylation changes are tissue-specific
with ELOVL2 promoter methylation as exception. Epigenet. Chromatin 2018, 11, 25. [CrossRef] [PubMed]
53. Zhu, Y.; Tchkonia, T.; Pirtskhalava, T.; Gower, A.C.; Ding, H.; Giorgadze, N.; Palmer, A.K.; Ikeno, Y.; Hubbard, G.B.; Lenburg,
M.; et al. The Achilles’ heel of senescent cells: From transcriptome to senolytic drugs. Aging Cell 2015, 14, 644–658. [CrossRef]
[PubMed]
54. Chaib, S.; Tchkonia, T.; Kirkland, J.L. Cellular senescence and senolytics: The path to the clinic. Nat. Med. 2022, 28, 1556–1568.
[CrossRef] [PubMed]
55. Aguayo-Mazzucato, C.; Andle, J.; Lee, T.B., Jr.; Midha, A.; Talemal, L.; Chipashvili, V.; Hollister-Lock, J.; van Deursen, J.; Weir, G.;
Bonner-Weir, S. Acceleration of beta Cell Aging Determines Diabetes and Senolysis Improves Disease Outcomes. Cell Metab. 2019,
30, 129–142.e4. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2024, 25, 643 17 of 21

56. Wang, L.; Wang, B.; Gasek, N.S.; Zhou, Y.; Cohn, R.L.; Martin, D.E.; Zuo, W.; Flynn, W.F.; Guo, C.; Jellison, E.R.; et al. Targeting
p21(Cip1) highly expressing cells in adipose tissue alleviates insulin resistance in obesity. Cell Metab. 2022, 34, 75–89.e8. [CrossRef]
[PubMed]
57. Saccon, T.D.; Nagpal, R.; Yadav, H.; Cavalcante, M.B.; Nunes, A.D.C.; Schneider, A.; Gesing, A.; Hughes, B.; Yousefzadeh, M.;
Tchkonia, T.; et al. Senolytic Combination of Dasatinib and Quercetin Alleviates Intestinal Senescence and Inflammation and
Modulates the Gut Microbiome in Aged Mice. J. Gerontol. A Biol. Sci. Med. Sci. 2021, 76, 1895–1905. [CrossRef] [PubMed]
58. Hense, J.D.; Garcia, D.N.; Isola, J.V.; Alvarado-Rincon, J.A.; Zanini, B.M.; Prosczek, J.B.; Stout, M.B.; Mason, J.B.; Walsh, P.T.;
Brieno-Enriquez, M.A.; et al. Senolytic treatment reverses obesity-mediated senescent cell accumulation in the ovary. Geroscience
2022, 44, 1747–1759. [CrossRef]
59. Alharbi, K.S.; Afzal, O.; Altamimi, A.S.A.; Almalki, W.H.; Kazmi, I.; Al-Abbasi, F.A.; Alzarea, S.I.; Makeen, H.A.; Albratty, M. A
study of the molecular mechanism of quercetin and dasatinib combination as senolytic in alleviating age-related and kidney
diseases. J. Food Biochem. 2022, 46, e14471. [CrossRef]
60. Murakami, T.; Inagaki, N.; Kondoh, H. Cellular Senescence in Diabetes Mellitus: Distinct Senotherapeutic Strategies for Adipose
Tissue and Pancreatic beta Cells. Front. Endocrinol. 2022, 13, 869414. [CrossRef]
61. Blagosklonny, M.V. Selective anti-cancer agents as anti-aging drugs. Cancer Biol. Ther. 2013, 14, 1092–1097. [CrossRef]
62. Zoncu, R.; Efeyan, A.; Sabatini, D.M. mTOR: From growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell
Biol. 2011, 12, 21–35. [CrossRef]
63. Bourgeois, B.; Madl, T. Regulation of cellular senescence via the FOXO4-p53 axis. FEBS Lett. 2018, 592, 2083–2097. [CrossRef]
[PubMed]
64. Park, H.K.; Yoon, N.G.; Lee, J.E.; Hu, S.; Yoon, S.; Kim, S.Y.; Hong, J.H.; Nam, D.; Chae, Y.C.; Park, J.B.; et al. Unleashing the full
potential of Hsp90 inhibitors as cancer therapeutics through simultaneous inactivation of Hsp90, Grp94, and TRAP1. Exp. Mol.
Med. 2020, 52, 79–91. [CrossRef]
65. Chen, D.D.; Peng, X.; Wang, Y.; Jiang, M.; Xue, M.; Shang, G.; Liu, X.; Jia, X.; Liu, B.; Lu, Y.; et al. HSP90 acts as a senomorphic
target in senescent retinal pigmental epithelial cells. Aging 2021, 13, 21547–21570. [CrossRef]
66. Zhu, Y.; Doornebal, E.J.; Pirtskhalava, T.; Giorgadze, N.; Wentworth, M.; Fuhrmann-Stroissnigg, H.; Niedernhofer, L.J.; Robbins,
P.D.; Tchkonia, T.; Kirkland, J.L. New agents that target senescent cells: The flavone, fisetin, and the BCL-X(L) inhibitors, A1331852
and A1155463. Aging 2017, 9, 955–963. [CrossRef] [PubMed]
67. Tao, Z.F.; Hasvold, L.; Wang, L.; Wang, X.; Petros, A.M.; Park, C.H.; Boghaert, E.R.; Catron, N.D.; Chen, J.; Colman, P.M.; et al.
Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity. ACS Med. Chem. Lett. 2014, 5, 1088–1093. [CrossRef]
[PubMed]
68. Wei, Y.; Zhang, L.; Wang, C.; Li, Z.; Luo, M.; Xie, G.; Yang, X.; Li, M.; Ren, S.; Zhao, D.; et al. Anti-apoptotic protein BCL-XL as a
therapeutic vulnerability in gastric cancer. Anim. Models Exp. Med. 2023, 6, 245–254. [CrossRef] [PubMed]
69. Moujalled, D.; Southon, A.G.; Saleh, E.; Brinkmann, K.; Ke, F.; Iliopoulos, M.; Cross, R.S.; Jenkins, M.R.; Nhu, D.; Wang, Z.; et al.
BH3 mimetic drugs cooperate with Temozolomide, JQ1 and inducers of ferroptosis in killing glioblastoma multiforme cells. Cell
Death Differ. 2022, 29, 1335–1348. [CrossRef]
70. Triana-Martinez, F.; Picallos-Rabina, P.; Da Silva-Alvarez, S.; Pietrocola, F.; Llanos, S.; Rodilla, V.; Soprano, E.; Pedrosa, P.; Ferreiros,
A.; Barradas, M.; et al. Identification and characterization of Cardiac Glycosides as senolytic compounds. Nat. Commun. 2019, 10,
4731. [CrossRef]
71. Seo, E.J.; Efferth, T.; Panossian, A. Curcumin downregulates expression of opioid-related nociceptin receptor gene (OPRL1) in
isolated neuroglia cells. Phytomedicine 2018, 50, 285–299. [CrossRef]
72. Lee, D.Y.; Lee, S.J.; Chandrasekaran, P.; Lamichhane, G.; O’Connell, J.F.; Egan, J.M.; Kim, Y. Dietary Curcumin Attenuates
Hepatic Cellular Senescence by Suppressing the MAPK/NF-kappaB Signaling Pathway in Aged Mice. Antioxidants 2023, 12, 1165.
[CrossRef] [PubMed]
73. Nam, S.; Kim, D.; Cheng, J.Q.; Zhang, S.; Lee, J.H.; Buettner, R.; Mirosevich, J.; Lee, F.Y.; Jove, R. Action of the Src family kinase
inhibitor, dasatinib (BMS-354825), on human prostate cancer cells. Cancer Res. 2005, 65, 9185–9189. [CrossRef] [PubMed]
74. Yousefzadeh, M.J.; Zhu, Y.; McGowan, S.J.; Angelini, L.; Fuhrmann-Stroissnigg, H.; Xu, M.; Ling, Y.Y.; Melos, K.I.; Pirtskhalava, T.;
Inman, C.L.; et al. Fisetin is a senotherapeutic that extends health and lifespan. EBioMedicine 2018, 36, 18–28. [CrossRef] [PubMed]
75. Huang, Y.; He, Y.; Makarcyzk, M.J.; Lin, H. Senolytic Peptide FOXO4-DRI Selectively Removes Senescent Cells From in vitro
Expanded Human Chondrocytes. Front. Bioeng. Biotechnol. 2021, 9, 677576. [CrossRef] [PubMed]
76. Fuhrmann-Stroissnigg, H.; Ling, Y.Y.; Zhao, J.; McGowan, S.J.; Zhu, Y.; Brooks, R.W.; Grassi, D.; Gregg, S.Q.; Stripay, J.L.;
Dorronsoro, A.; et al. Identification of HSP90 inhibitors as a novel class of senolytics. Nat. Commun. 2017, 8, 422. [CrossRef]
[PubMed]
77. Iida, K.; Naiki, T.; Naiki-Ito, A.; Suzuki, S.; Kato, H.; Nozaki, S.; Nagai, T.; Etani, T.; Nagayasu, Y.; Ando, R.; et al. Luteolin
suppresses bladder cancer growth via regulation of mechanistic target of rapamycin pathway. Cancer Sci. 2020, 111, 1165–1179.
[CrossRef] [PubMed]
78. Takaya, K.; Ishii, T.; Asou, T.; Kishi, K. Navitoclax (ABT-263) Rejuvenates Human Skin by Eliminating Senescent Dermal
Fibroblasts in a Mouse/Human Chimeric Model. Rejuvenation Res. 2023, 26, 9–20. [CrossRef] [PubMed]
79. Chung, H.; Kim, C. Nutlin-3a for age-related macular degeneration. Aging 2022, 14, 5614–5616. [CrossRef]
Int. J. Mol. Sci. 2024, 25, 643 18 of 21

80. Wang, Y.; Chang, J.; Liu, X.; Zhang, X.; Zhang, S.; Zhang, X.; Zhou, D.; Zheng, G. Discovery of piperlongumine as a potential
novel lead for the development of senolytic agents. Aging 2016, 8, 2915–2926. [CrossRef]
81. Wang, R.; Yu, Z.; Sunchu, B.; Shoaf, J.; Dang, I.; Zhao, S.; Caples, K.; Bradley, L.; Beaver, L.M.; Ho, E.; et al. Rapamycin inhibits the
secretory phenotype of senescent cells by a Nrf2-independent mechanism. Aging Cell 2017, 16, 564–574. [CrossRef]
82. Powers, M.V.; Valenti, M.; Miranda, S.; Maloney, A.; Eccles, S.A.; Thomas, G.; Clarke, P.A.; Workman, P. Mode of cell death
induced by the HSP90 inhibitor 17-AAG (tanespimycin) is dependent on the expression of pro-apoptotic BAX. Oncotarget 2013, 4,
1963–1975. [CrossRef] [PubMed]
83. Harris, D.T.; Hilgaertner, J.; Simonson, C.; Ablin, R.J.; Badowski, M. Cell-based therapy for epithelial wounds. Cytotherapy 2012,
14, 802–810. [CrossRef] [PubMed]
84. Pippias, M.; Jager, K.J.; Asberg, A.; Berger, S.P.; Finne, P.; Heaf, J.G.; Kerschbaum, J.; Lempinen, M.; Magaz, A.; Massy, Z.A.; et al.
Young deceased donor kidneys show a survival benefit over older donor kidneys in transplant recipients aged 20-50 years: A
study by the ERA-EDTA Registry. Nephrol. Dial. Transplant. 2020, 35, 534–543. [CrossRef] [PubMed]
85. Iske, J.; Matsunaga, T.; Zhou, H.; Tullius, S.G. Donor and Recipient Age-Mismatches: The Potential of Transferring Senescence.
Front. Immunol. 2021, 12, 671479. [CrossRef] [PubMed]
86. Keren, A.; Bertolini, M.; Keren, Y.; Ullmann, Y.; Paus, R.; Gilhar, A. Human organ rejuvenation by VEGF-A: Lessons from the skin.
Sci. Adv. 2022, 8, eabm6756. [CrossRef] [PubMed]
87. Wang, L.; Wei, J.; Da Fonseca Ferreira, A.; Wang, H.; Zhang, L.; Zhang, Q.; Bellio, M.A.; Chu, X.M.; Khan, A.; Jayaweera, D.; et al.
Rejuvenation of Senescent Endothelial Progenitor Cells by Extracellular Vesicles Derived From Mesenchymal Stromal Cells. JACC
Basic Transl. Sci. 2020, 5, 1127–1141. [CrossRef]
88. Grigorian-Shamagian, L.; Liu, W.; Fereydooni, S.; Middleton, R.C.; Valle, J.; Cho, J.H.; Marban, E. Cardiac and systemic
rejuvenation after cardiosphere-derived cell therapy in senescent rats. Eur. Heart J. 2017, 38, 2957–2967. [CrossRef] [PubMed]
89. Brunet, A.; Goodell, M.A.; Rando, T.A. Ageing and rejuvenation of tissue stem cells and their niches. Nat. Rev. Mol. Cell Biol. 2023,
24, 45–62. [CrossRef]
90. Zhang, D.Y.; Zhang, C.F.; Fu, B.C.; Sun, L.; Wang, X.Q.; Chen, W.; Liu, W.; Liu, K.Y.; Du, G.Q.; Ma, C.Y.; et al. Sirtuin3 protects
aged human mesenchymal stem cells against oxidative stress and enhances efficacy of cell therapy for ischaemic heart diseases. J.
Cell Mol. Med. 2018, 22, 5504–5517. [CrossRef]
91. Chulpanova, D.S.; Kitaeva, K.V.; Tazetdinova, L.G.; James, V.; Rizvanov, A.A.; Solovyeva, V.V. Application of Mesenchymal Stem
Cells for Therapeutic Agent Delivery in Anti-tumor Treatment. Front. Pharmacol. 2018, 9, 259. [CrossRef]
92. Awad, M.E.; Hussein, K.A.; Helwa, I.; Abdelsamid, M.F.; Aguilar-Perez, A.; Mohsen, I.; Hunter, M.; Hamrick, M.W.; Isales, C.M.;
Elsalanty, M.; et al. Meta-Analysis and Evidence Base for the Efficacy of Autologous Bone Marrow Mesenchymal Stem Cells
in Knee Cartilage Repair: Methodological Guidelines and Quality Assessment. Stem Cells Int. 2019, 2019, 3826054. [CrossRef]
[PubMed]
93. Gasanova, S.Y. Cell therapy for destructive pancreatitis. Khirurgiia 2022, 9, 50–55. [CrossRef]
94. Gjerde, C.; Mustafa, K.; Hellem, S.; Rojewski, M.; Gjengedal, H.; Yassin, M.A.; Feng, X.; Skaale, S.; Berge, T.; Rosen, A.; et al. Cell
therapy induced regeneration of severely atrophied mandibular bone in a clinical trial. Stem Cell Res. Ther. 2018, 9, 213. [CrossRef]
[PubMed]
95. Zarei, F.; Abbaszadeh, A. Application of Cell Therapy for Anti-Aging Facial Skin. Curr. Stem Cell Res. Ther. 2019, 14, 244–248.
[CrossRef] [PubMed]
96. Zhang, X.; Liu, T.; Hou, X.; Zhou, Z.; Zhang, F.; Ma, H.; Wu, X.; Jiang, J. Exosomes secreted by mesenchymal stem cells delay
brain aging by upregulating SIRT1 expression. Sci. Rep. 2023, 13, 13213. [CrossRef] [PubMed]
97. Zhang, D.Y.; Gao, T.; Xu, R.J.; Sun, L.; Zhang, C.F.; Bai, L.; Chen, W.; Liu, K.Y.; Zhou, Y.; Jiao, X.; et al. SIRT3 Transfection of Aged
Human Bone Marrow-Derived Mesenchymal Stem Cells Improves Cell Therapy-Mediated Myocardial Repair. Rejuvenation Res.
2020, 23, 453–464. [CrossRef]
98. Primorac, D.; Molnar, V.; Rod, E.; Jelec, Z.; Cukelj, F.; Matisic, V.; Vrdoljak, T.; Hudetz, D.; Hajsok, H.; Boric, I. Knee Osteoarthritis:
A Review of Pathogenesis and State-of-the-Art Non-Operative Therapeutic Considerations. Genes 2020, 11, 854. [CrossRef]
99. Andia, I.; Maffulli, N.; Burgos-Alonso, N. Stromal vascular fraction technologies and clinical applications. Expert. Opin. Biol. Ther.
2019, 19, 1289–1305. [CrossRef]
100. Rowe, G.; Tracy, E.; Beare, J.E.; LeBlanc, A.J. Cell therapy rescues aging-induced beta-1 adrenergic receptor and GRK2 dysfunction
in the coronary microcirculation. Geroscience 2022, 44, 329–348. [CrossRef]
101. Mautner, K.; Bowers, R.; Easley, K.; Fausel, Z.; Robinson, R. Functional Outcomes Following Microfragmented Adipose Tissue
Versus Bone Marrow Aspirate Concentrate Injections for Symptomatic Knee Osteoarthritis. Stem Cells Transl. Med. 2019, 8,
1149–1156. [CrossRef]
102. Weng, H.P.; Cheng, Y.Y.; Lee, H.L.; Hsu, T.Y.; Chang, Y.T.; Shen, Y.A. Enhanced Platelet-Rich Plasma (ePRP) Stimulates Wound
Healing through Effects on Metabolic Reprogramming in Fibroblasts. Int. J. Mol. Sci. 2021, 22, 12623. [CrossRef] [PubMed]
103. Wu, Q.; Luo, X.; Xiong, Y.; Liu, G.; Wang, J.; Chen, X.; Mi, B. Platelet-rich plasma versus hyaluronic acid in knee osteoarthritis: A
meta-analysis with the consistent ratio of injection. J. Orthop. Surg. 2020, 28, 2309499019887660. [CrossRef] [PubMed]
104. Grether-Beck, S.; Marini, A.; Jaenicke, T.; Goessens-Ruck, P.; McElwee, K.J.; Hoffmann, R.; Krutmann, J. Autologous Cell Therapy
for Aged Human Skin: A Randomized, Placebo-Controlled, Phase-I Study. Skin Pharmacol. Physiol. 2020, 33, 9–16. [CrossRef]
[PubMed]
Int. J. Mol. Sci. 2024, 25, 643 19 of 21

105. Sugita, S.; Iwasaki, Y.; Makabe, K.; Kamao, H.; Mandai, M.; Shiina, T.; Ogasawara, K.; Hirami, Y.; Kurimoto, Y.; Takahashi, M.
Successful Transplantation of Retinal Pigment Epithelial Cells from MHC Homozygote iPSCs in MHC-Matched Models. Stem
Cell Rep. 2016, 7, 635–648. [CrossRef] [PubMed]
106. Yang, J.M.; Chung, S.; Yun, K.; Kim, B.; So, S.; Kang, S.; Kang, E.; Lee, J.Y. Long-term effects of human induced pluripotent stem
cell-derived retinal cell transplantation in Pde6b knockout rats. Exp. Mol. Med. 2021, 53, 631–642. [CrossRef] [PubMed]
107. Sugita, S.; Futatsugi, Y.; Ishida, M.; Edo, A.; Takahashi, M. Retinal Pigment Epithelial Cells Derived from Induced Pluripotent
Stem (iPS) Cells Suppress or Activate T Cells via Costimulatory Signals. Int. J. Mol. Sci. 2020, 21, 6507. [CrossRef] [PubMed]
108. Zhu, D.; Xie, M.; Gademann, F.; Cao, J.; Wang, P.; Guo, Y.; Zhang, L.; Su, T.; Zhang, J.; Chen, J. Protective effects of human
iPS-derived retinal pigmented epithelial cells on retinal degenerative disease. Stem Cell Res. Ther. 2020, 11, 98. [CrossRef]
109. Martin-Lopez, M.; Gonzalez-Munoz, E.; Gomez-Gonzalez, E.; Sanchez-Pernaute, R.; Marquez-Rivas, J.; Fernandez-Munoz, B.
Modeling chronic cervical spinal cord injury in aged rats for cell therapy studies. J. Clin. Neurosci. 2021, 94, 76–85. [CrossRef]
110. Gargioli, C.; Coletta, M.; De Grandis, F.; Cannata, S.M.; Cossu, G. PlGF-MMP-9-expressing cells restore microcirculation and
efficacy of cell therapy in aged dystrophic muscle. Nat. Med. 2008, 14, 973–978. [CrossRef]
111. Amor, C.; Feucht, J.; Leibold, J.; Ho, Y.J.; Zhu, C.; Alonso-Curbelo, D.; Mansilla-Soto, J.; Boyer, J.A.; Li, X.; Giavridis, T.; et al.
Senolytic CAR T cells reverse senescence-associated pathologies. Nature 2020, 583, 127–132. [CrossRef]
112. Liu, D.; Zhu, M.; Zhang, Y.; Diao, Y. Crossing the blood-brain barrier with AAV vectors. Metab. Brain Dis. 2021, 36, 45–52.
[CrossRef]
113. Issa, S.S.; Shaimardanova, A.A.; Solovyeva, V.V.; Rizvanov, A.A. Various AAV Serotypes and Their Applications in Gene Therapy:
An Overview. Cells 2023, 12, 785. [CrossRef] [PubMed]
114. Shaimardanova, A.A.; Chulpanova, D.S.; Mullagulova, A.I.; Afawi, Z.; Gamirova, R.G.; Solovyeva, V.V.; Rizvanov, A.A. Gene and
Cell Therapy for Epilepsy: A Mini Review. Front. Mol. Neurosci. 2022, 15, 868531. [CrossRef] [PubMed]
115. Yu, J.; Li, T.; Zhu, J. Gene Therapy Strategies Targeting Aging-Related Diseases. Aging Dis. 2023, 14, 398–417. [CrossRef]
116. Shaimardanova, A.A.; Chulpanova, D.S.; Solovyeva, V.V.; Issa, S.S.; Mullagulova, A.I.; Titova, A.A.; Mukhamedshina, Y.O.;
Timofeeva, A.V.; Aimaletdinov, A.M.; Nigmetzyanov, I.R.; et al. Increasing beta-hexosaminidase A activity using genetically
modified mesenchymal stem cells. Neural Regen. Res. 2024, 19, 212–219. [CrossRef] [PubMed]
117. Bijlani, S.; Pang, K.M.; Sivanandam, V.; Singh, A.; Chatterjee, S. The Role of Recombinant AAV in Precise Genome Editing. Front.
Genome Ed. 2021, 3, 799722. [CrossRef]
118. Vila, L.; Roca, C.; Elias, I.; Casellas, A.; Lage, R.; Franckhauser, S.; Bosch, F. AAV-mediated Sirt1 overexpression in skeletal muscle
activates oxidative capacity but does not prevent insulin resistance. Mol. Ther. Methods Clin. Dev. 2016, 5, 16072. [CrossRef]
[PubMed]
119. Adu-Agyeiwaah, Y.; Vieira, C.P.; Asare-Bediako, B.; Li Calzi, S.; DuPont, M.; Floyd, J.; Boye, S.; Chiodo, V.; Busik, J.V.; Grant, M.B.
Intravitreal Administration of AAV2-SIRT1 Reverses Diabetic Retinopathy in a Mouse Model of Type 2 Diabetes. Transl. Vis. Sci.
Technol. 2023, 12, 20. [CrossRef]
120. Gao, P.; You, M.; Li, L.; Zhang, Q.; Fang, X.; Wei, X.; Zhou, Q.; Zhang, H.; Wang, M.; Lu, Z.; et al. Salt-Induced Hepatic
Inflammatory Memory Contributes to Cardiovascular Damage Through Epigenetic Modulation of SIRT3. Circulation 2022, 145,
375–391. [CrossRef]
121. Diao, Z.; Ji, Q.; Wu, Z.; Zhang, W.; Cai, Y.; Wang, Z.; Hu, J.; Liu, Z.; Wang, Q.; Bi, S.; et al. SIRT3 consolidates heterochromatin and
counteracts senescence. Nucleic Acids Res. 2021, 49, 4203–4219. [CrossRef]
122. Ling, W.; Krager, K.; Richardson, K.K.; Warren, A.D.; Ponte, F.; Aykin-Burns, N.; Manolagas, S.C.; Almeida, M.; Kim, H.N.
Mitochondrial Sirt3 contributes to the bone loss caused by aging or estrogen deficiency. JCI Insight 2021, 6, e146728. [CrossRef]
123. Song, S.; Ding, Y.; Dai, G.L.; Zhang, Y.; Xu, M.T.; Shen, J.R.; Chen, T.T.; Chen, Y.; Meng, G.L. Sirtuin 3 deficiency exacerbates
diabetic cardiomyopathy via necroptosis enhancement and NLRP3 activation. Acta Pharmacol. Sin. 2021, 42, 230–241. [CrossRef]
[PubMed]
124. Li, G.; Qin, H.; Zhou, M.; Zhang, T.; Zhang, Y.; Ding, H.; Xu, L.; Song, J. Knockdown of SIRT3 perturbs protective effects of irisin
against bone loss in diabetes and periodontitis. Free Radic. Biol. Med. 2023, 200, 11–25. [CrossRef] [PubMed]
125. Grootaert, M.O.J.; Finigan, A.; Figg, N.L.; Uryga, A.K.; Bennett, M.R. SIRT6 Protects Smooth Muscle Cells From Senescence and
Reduces Atherosclerosis. Circ. Res. 2021, 128, 474–491. [CrossRef] [PubMed]
126. Yang, Z.; Huang, Y.; Zhu, L.; Yang, K.; Liang, K.; Tan, J.; Yu, B. SIRT6 promotes angiogenesis and hemorrhage of carotid plaque
via regulating HIF-1alpha and reactive oxygen species. Cell Death Dis. 2021, 12, 77. [CrossRef] [PubMed]
127. Turner, K.J.; Vasu, V.; Griffin, D.K. Telomere Biology and Human Phenotype. Cells 2019, 8, 73. [CrossRef] [PubMed]
128. Jaijyan, D.K.; Selariu, A.; Cruz-Cosme, R.; Tong, M.; Yang, S.; Stefa, A.; Kekich, D.; Sadoshima, J.; Herbig, U.; Tang, Q.; et al. New
intranasal and injectable gene therapy for healthy life extension. Proc. Natl. Acad. Sci. USA 2022, 119, e2121499119. [CrossRef]
129. Miller, J.D.; Ganat, Y.M.; Kishinevsky, S.; Bowman, R.L.; Liu, B.; Tu, E.Y.; Mandal, P.K.; Vera, E.; Shim, J.W.; Kriks, S.; et al. Human
iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell 2013, 13, 691–705. [CrossRef]
130. Li, Z.; Zhang, Y.; Sui, S.; Hua, Y.; Zhao, A.; Tian, X.; Wang, R.; Guo, W.; Yu, W.; Zou, K.; et al. Targeting HMGB3/hTERT axis for
radioresistance in cervical cancer. J. Exp. Clin. Cancer Res. 2020, 39, 243. [CrossRef]
131. Yamazaki, Y.; Imura, A.; Urakawa, I.; Shimada, T.; Murakami, J.; Aono, Y.; Hasegawa, H.; Yamashita, T.; Nakatani, K.; Saito, Y.;
et al. Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho
levels in healthy subjects. Biochem. Biophys. Res. Commun. 2010, 398, 513–518. [CrossRef]
Int. J. Mol. Sci. 2024, 25, 643 20 of 21

132. Gao, D.; Wang, S.; Lin, Y.; Sun, Z. In vivo AAV delivery of glutathione reductase gene attenuates anti-aging gene klotho
deficiency-induced kidney damage. Redox Biol. 2020, 37, 101692. [CrossRef]
133. Chen, K.; Wang, S.; Sun, Z. In Vivo Cardiac-specific Expression of Adenylyl Cyclase 4 Gene Protects against Klotho Deficiency-
induced Heart Failure. Transl. Res. 2022, 244, 101–113. [CrossRef] [PubMed]
134. Roig-Soriano, J.; Sanchez-de-Diego, C.; Esandi-Jauregui, J.; Verdes, S.; Abraham, C.R.; Bosch, A.; Ventura, F.; Chillon, M.
Differential toxicity profile of secreted and processed alpha-Klotho expression over mineral metabolism and bone microstructure.
Sci. Rep. 2023, 13, 4211. [CrossRef] [PubMed]
135. Chuchana, P.; Mausset-Bonnefont, A.L.; Mathieu, M.; Espinoza, F.; Teigell, M.; Toupet, K.; Ripoll, C.; Djouad, F.; Noel, D.;
Jorgensen, C.; et al. Secreted alpha-Klotho maintains cartilage tissue homeostasis by repressing NOS2 and ZIP8-MMP13 catabolic
axis. Aging 2018, 10, 1442–1453. [CrossRef] [PubMed]
136. Xiang, T.; Luo, X.; Zeng, C.; Li, S.; Ma, M.; Wu, Y. Klotho ameliorated cognitive deficits in a temporal lobe epilepsy rat model by
inhibiting ferroptosis. Brain Res. 2021, 1772, 147668. [CrossRef] [PubMed]
137. Xiang, T.; Luo, X.; Ye, L.; Huang, H.; Wu, Y. Klotho alleviates NLRP3 inflammasome-mediated neuroinflammation in a temporal
lobe epilepsy rat model by activating the Nrf2 signaling pathway. Epilepsy Behav. 2022, 128, 108509. [CrossRef] [PubMed]
138. Wang, Y.; Sun, Z. Klotho gene delivery prevents the progression of spontaneous hypertension and renal damage. Hypertension
2009, 54, 810–817. [CrossRef] [PubMed]
139. Roig-Soriano, J.; Grinan-Ferre, C.; Espinosa-Parrilla, J.F.; Abraham, C.R.; Bosch, A.; Pallas, M.; Chillon, M. AAV-mediated
expression of secreted and transmembrane alphaKlotho isoforms rescues relevant aging hallmarks in senescent SAMP8 mice.
Aging Cell 2022, 21, e13581. [CrossRef]
140. Fan, J.; Wang, S.; Chen, K.; Sun, Z. Aging impairs arterial compliance via Klotho-mediated downregulation of B-cell population
and IgG levels. Cell Mol. Life Sci. 2022, 79, 494. [CrossRef]
141. Sewell, P.E.; Ediriweera, D.; Gomez Rios, E.; Guadarrama, O.A.; Eusebio, Y.; Gonzalez, L.; Parrish, E.L. Safety Study of AAV hTert
and Klotho Gene Transfer Therapy for Dementia. J. Regen. Biol. Med. 2021, 3, 1–15. [CrossRef]
142. Clemens, Z.; Sivakumar, S.; Pius, A.; Sahu, A.; Shinde, S.; Mamiya, H.; Luketich, N.; Cui, J.; Dixit, P.; Hoeck, J.D.; et al. The biphasic
and age-dependent impact of klotho on hallmarks of aging and skeletal muscle function. eLife 2021, 10, e61138. [CrossRef]
[PubMed]
143. Jimenez, V.; Jambrina, C.; Casana, E.; Sacristan, V.; Munoz, S.; Darriba, S.; Rodo, J.; Mallol, C.; Garcia, M.; Leon, X.; et al. FGF21
gene therapy as treatment for obesity and insulin resistance. EMBO Mol. Med. 2018, 10, e8791. [CrossRef] [PubMed]
144. Youm, Y.H.; Horvath, T.L.; Mangelsdorf, D.J.; Kliewer, S.A.; Dixit, V.D. Prolongevity hormone FGF21 protects against immune
senescence by delaying age-related thymic involution. Proc. Natl. Acad. Sci. USA 2016, 113, 1026–1031. [CrossRef] [PubMed]
145. Villarroya, J.; Gallego-Escuredo, J.M.; Delgado-Angles, A.; Cairo, M.; Moure, R.; Gracia Mateo, M.; Domingo, J.C.; Domingo, P.;
Giralt, M.; Villarroya, F. Aging is associated with increased FGF21 levels but unaltered FGF21 responsiveness in adipose tissue.
Aging Cell 2018, 17, e12822. [CrossRef] [PubMed]
146. Davidsohn, N.; Pezone, M.; Vernet, A.; Graveline, A.; Oliver, D.; Slomovic, S.; Punthambaker, S.; Sun, X.; Liao, R.; Bonventre, J.V.;
et al. A single combination gene therapy treats multiple age-related diseases. Proc. Natl. Acad. Sci. USA 2019, 116, 23505–23511.
[CrossRef] [PubMed]
147. Grunewald, M.; Kumar, S.; Sharife, H.; Volinsky, E.; Gileles-Hillel, A.; Licht, T.; Permyakova, A.; Hinden, L.; Azar, S.; Friedmann,
Y.; et al. Counteracting age-related VEGF signaling insufficiency promotes healthy aging and extends life span. Science 2021,
373, eabc8479. [CrossRef]
148. Qi, X.; Francelin, C.; Mitter, S.; Boye, S.L.; Gu, H.; Quigley, J.; Grant, M.B.; Boulton, M.E. beta-secretase 1 overexpression by
AAV-mediated gene delivery prevents retina degeneration in a mouse model of age-related macular degeneration. Mol. Ther.
2023, 31, 2042–2055. [CrossRef]
149. Logan, C.; Lyzogubov, V.; Bora, N.; Bora, P. Role of Adiponectin Peptide I (APNp1) in Age-Related Macular Degeneration.
Biomolecules 2022, 12, 1232. [CrossRef]
150. Choudhary, M.; Ildefonso, C.J.; Lewin, A.S.; Malek, G. Gene Delivery of a Caspase Activation and Recruitment Domain Improves
Retinal Pigment Epithelial Function and Modulates Inflammation in a Mouse Model with Features of Dry Age-Related Macular
Degeneration. J. Ocul. Pharmacol. Ther. 2022, 38, 359–371. [CrossRef]
151. Millington-Ward, S.; Chadderton, N.; Finnegan, L.K.; Post, I.J.M.; Carrigan, M.; Gardiner, T.; Peixoto, E.; Maloney, D.; Humphries,
M.M.; Stitt, A.; et al. AAV-mediated gene therapy improving mitochondrial function provides benefit in age-related macular
degeneration models. Clin. Transl. Med. 2022, 12, e952. [CrossRef]
152. Molinari, C.; Morsanuto, V.; Ruga, S.; Notte, F.; Farghali, M.; Galla, R.; Uberti, F. The Role of BDNF on Aging-Modulation Markers.
Brain Sci. 2020, 10, 285. [CrossRef] [PubMed]
153. Chen, F.; Swartzlander, D.B.; Ghosh, A.; Fryer, J.D.; Wang, B.; Zheng, H. Clusterin secreted from astrocyte promotes excitatory
synaptic transmission and ameliorates Alzheimer’s disease neuropathology. Mol. Neurodegener. 2021, 16, 5. [CrossRef] [PubMed]
154. Bartus, R.T.; Baumann, T.L.; Brown, L.; Kruegel, B.R.; Ostrove, J.M.; Herzog, C.D. Advancing neurotrophic factors as treatments
for age-related neurodegenerative diseases: Developing and demonstrating “clinical proof-of-concept” for AAV-neurturin
(CERE-120) in Parkinson’s disease. Neurobiol. Aging 2013, 34, 35–61. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2024, 25, 643 21 of 21

155. Zuo, W.; Zhao, J.; Zhang, J.; Fang, Z.; Deng, J.; Fan, Z.; Guo, Y.; Han, J.; Hou, W.; Dong, H.; et al. MD2 contributes to
the pathogenesis of perioperative neurocognitive disorder via the regulation of alpha5GABA(A) receptors in aged mice. J.
Neuroinflamm. 2021, 18, 204. [CrossRef]
156. Klein, R.L.; Hirko, A.C.; Meyers, C.A.; Grimes, J.R.; Muzyczka, N.; Meyer, E.M. NGF gene transfer to intrinsic basal forebrain
neurons increases cholinergic cell size and protects from age-related, spatial memory deficits in middle-aged rats. Brain Res. 2000,
875, 144–151. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.