INTRODUCTION

Human gene therapy is defined as the treatment of disorder or disease through

transfer of engineered genetic material into human cells, often by viral
transduction. Since the introduction of science fiction, the popular press has
toyed with the notion of viral gene delivery and its terrifying implications. One
of the more recent popular works on the topic is the 2007 remake of Richard
Matheson’s classic 1954 novel I Am Legend, which details events following
the discovery, release, and mutation of a genetically re-engineered measles virus
that was initially hailed as the cure for cancer. This adapted novel, which has
been redone in three instances as a feature film, outlines the seemingly
inevitable worldwide destruction that could result from viral gene therapy.

With an emotionally stirring history of

fictional violence and a debate that provokes both moral and medical issues, it
may be surprising that, since 1990, billions of dollars have been spent on
hundreds of human viral gene therapy clinical trials. Our society is in the midst
of a paradigm shift that began with the discovery of viruses as dangerous
infectious agents and will end with the use of viruses to cure disease and
regenerate tissues.On January 19, 1989, the director of the National Institutes of
Health (NIH), Dr. James A. Wyngaarden, approved the first clinical protocol to
insert a foreign gene into the immune cells of persons with cancer. On
September 14, 1990, W. French Anderson and his colleagues at the NIH
performed the first approved gene therapy procedure on a four-year-old girl
born with severe combined immunodeficiency (SCID). Despite the viral horror
stories written by the popular media, this initial trial was largely a success, and
the most recent report on this individual in 2004 noted that she is thriving as an
18-year-old teenager in suburban Cleveland. Over the next ten years, 300
clinical gene therapy trials were performed on about 3000 individuals. The field
was then blackened with the death of an 18-year-old male four days after the
introduction of 38 trillion particles of recombinant adenovirus into his liver.
Despite this tragedy, we continue to move forward because of the great promise
of novel genetic treatments that, when perfected, will likely outshine current
methods, such as protein therapy or pharmacotherapeutics, for treatment of
many diseases and defects.We are now nearing the 20-year mark since the first
gene therapy trial. Though success has been limited, the future still seems
overwhelmingly promising, and we are steadily approaching an acceptable
safety record. This review will explore non-viral and viral methods available for
transgene introduction as well as their current and potential applications for
craniofacial regeneration and therapy, with emphasis on future development and
design.

NON VIRAL GENE THERAPY

Though this review will focus mostly on viral methods of gene delivery, it is
essential to recognize that many advances have been made in the field of non-
viral gene therapy. Polymeric gene delivery is desired because of its relative
safety, low immunogenicity and toxicity, ease of administration and
manufacture, and lack of DNA insert size limitation. The main disadvantage is
insufficient gene transfer efficiency due to the need for post-uptake endosomal
escape and nuclear translocation of the DNA complex. In this respect, clinical
efficiency and specificity standards have not yet been met.

VIRAL GENE DELIVERY

Viruses have undergone millions of years of evolution and are a species-
conserved way of introducing DNA to cells . Scientists are now attempting to
fine-tune these gene delivery vehicles for treatment of human disease and
defects. Regardless of method selection, there are three universal requirements
for viral gene therapy vectors. First, the delivery system must be safe and
immunologically inert. Second, it must protect the genetic material from
degradation. Third, the vector must encode an effective therapeutic gene that
has sustained expression at a defined target site. For true commercial
application, the packaged vector must also be easily produced and processed
and have a reasonable shelf-life. As we near the 20-year mark from the first
human gene therapy clinical trial, significant advances have been made in
satisfying these three requirements. However, new objectives—such as tissue-
specific targeting, site-specific chromosomal integration, and controlled
infection of both dividing and non-dividing cells—have emerged. Though
negative publicity has attached a significant stigma to viral gene therapy, it is
indeed the most efficient method of gene transfer, and basic research and
clinical trials are rapidly moving to overturn the safety concerns.Our society is
in the midst of a paradigm shift that began with the discovery of viruses as
dangerous infectious agents and will end with the use of viruses to cure disease
and regenerate tissues. However, safety concerns still limit the universal
acceptance of this strategy. These concerns include the accidental generation of
replication competent viruses during vector production and the packaging or
mobilization of the engineered vector by endogenous retroviruses present in the
human genome. Either of these could lead to horizontal dissemination of new
viruses from gene therapy patients. Localized concerns include random
insertion or mutagenesis of the vector leading to cancer, or germ cell alteration
resulting in vertical inheritance of the acquired gene. The need for controlled
genome integration hit home when two of the 11 persons treated for X-SCID
with retrovirally transduced stem cells developed leukemia due to insertion of
the transgene near the oncogenic gene LMO2. Site-specific chromosomal
integration, conditional expression of the transgene only in target cells, and the

use of self-inactivating (SIN) retroviral vectors have been proposed and may
significantly improve the safety of viral therapy. The following sections will
review the use of viral vectors for in vivo therapy, emphasizing the construction
and advantages of different viruses.
CLINICAL GENE THERAPY
There are three main strategies for gene delivery: in vivo, in vitro, and ex vivo.
Though the most direct method is in vivo injection, this approach lacks the
improved patient safety of in vitro and ex vivo methods. Systemic delivery is
desirable if the target tissue is not directly accessible. However, this method
often results in low specificity of gene expression, risks of toxicity due to the
high vector concentration required, and potential damage to the function of
healthy tissues. Alternatively, matrix-based delivery allows for tissue-specific
gene delivery, higher localized loading of DNA or virus, and increased control
over the structural microenvironment. Thus far, human in vivo clinical trials
have introduced adenovirus, AAV, retrovirus, and herpes simplex virus by
intravenous (IV) injection, intra-tissue injection, or lung aerosol.

In contrast, ex vivo trials have focused on stable retroviral transduction of

rapidly dividing populations such as CD8+ T-cells, hematopoietic stem cells,
hepatocytes, and fibroblasts, followed by IV or local re-introduction. At the
time of this publication, a search of the NIH Genetic Modification Clinical
Research Information System (GeMCRIS) revealed 908 total gene therapy
clinical trial entries in the database. At clinicaltrials.gov, a search for
interventions with “gene transfer” OR “gene therapy” returned 174 studies, of
which 145 are viral-based, with 84 active, 48 completed, and 7 terminated. This
cross-section of results translates to 1605 persons who have participated in this
subset of completed gene therapy trials and nearly 5000 total active or
anticipated participants, based on each study’s documented enrollment since
1990. The following sections will briefly review the progress of gene therapy
since 1990.

GENE EDITION
The concept of gene therapy is to fix a genetic problem at its source. If, for
instance, a mutation in a certain gene causes the production of a dysfunctional
protein resulting (usually recessively) in an inherited disease, gene therapy
could be used to deliver a copy of this gene that does not contain the deleterious
mutation and thereby produces a functional protein. This strategy is referred to
as gene replacement therapy and could be employed to treat inherited retinal
diseases.
While the concept of gene replacement therapy is mostly suitable for recessive
diseases, novel strategies have been suggested that are capable of also treating
conditions with a dominant pattern of inheritane

 The introduction of CRISPR gene editing has opened new doors for its
application and utilization in gene therapy, as instead of pure replacement of
a gene, it enables correction of the particular genetic defect. [40] Solutions to
medical hurdles, such as the eradication of latent human immunodeficiency
virus (HIV) reservoirs and correction of the mutation that causes sickle cell
disease, may be available as a therapeutic option in the future. [67][68][69]
 Prosthetic gene therapy aims to enable cells of the body to take over
functions they physiologically do not carry out. One example is the so-called
vision restoration gene therapy, that aims to restore vision in patients with
end-stage retinal diseases.[70][71] In end-stage retinal diseases, the
photoreceptors, as the primary light sensitive cells of the retina are
irreversibly lost. By the means of prosthetic gene therapy light sensitive
proteins are delivered into the remaining cells of the retina, to render them
light sensitive and thereby enable them to signal visual information towards
the brain
In vivo, gene editing systems using CRISPR have been used in studies with
mice to treat cancer and have been effective at reducing tumors. [72]: 18 In vitro,
the CRISPR system has been used to treat HPV cancer tumors. Adeno-
associated virus, Lentivirus based vectors have been to introduce the genome
for the CRISPR system.

TREATMENT OF DISEASE
Gene therapy is specially suited for long-term delivery of a transgene to persons
with a single genetic deficiency that is not amenable to protein or
pharmacokinetic therapy. This was the premise of the first successful gene
therapy clinical trials that inserted genes ex vivo into CD34+ cells to treat
persons with SCID. Amazingly, persistence of the adenosine deaminase (ADA)
transgene was noted in peripheral blood leukocytes 12 yrs post-therapy without
adverse events. Since 1990, clinical treatment of genetic diseases—including
cystic fibrosis, hemophilia, Leber congenital amaurosis, muscular dystrophy,
ornithine transcarbamylase deficiency, Pompe disease, and Gaucher’s disease—
has been attempted, with promising documented success. Following the SCID
trials, treatment of cystic fibrosis by re-introduction of the cystic fibrosis
transmembrane regulator (CFTR) chloride ion channel to lung epithelial cells
was highly targeted and was the first use of rAAV in humans. However, like

many other in vivo and ex vivo clinical trials, transduction efficiency was
generally insufficient to improve clinical parameters significantly. Apart from
SCID, the most promising documented results for genetic deficiency correction
have been the replacement of factor IX (F-IX) in hemophilia. Studies by Avigen
Inc. have examined rAAV2-mediated F-IX delivery to the liver. In dogs,
therapeutic levels of F-IX were achieved for multiple years following vector
treatment. In humans, delivery of rAAV2.F-IX through the hepatic artery
achieved therapeutic levels of F-IX expression for approximately 8 wks. It
appears that cell-mediated immunity to the rAAV2 capsid limits expression in
humans. Thus, immunomodulation and capsid engineering may make F-IX
therapy a near-future reality. Gene therapy is also highly desired for the
treatment of neurologic and other chronic disease. Clinical trials have been
implemented and/or completed for the treatment of HIV/AIDS, arthritis, angina
pectoris, solid tumors, Parkinson’s disease, Huntington’s disease, Alzheimer’s
disease, Batten disease, Canavan disease, and familial hypercholesterolemia.
Despite the many hurdles, most clinical trials are progressing steadily, with
treatments for angina pectoris, prostate cancer, non-small-cell lung cancer, and
head and neck cancer now entering phase III clinical trials.

Gene Therapy For Craniofacial

Regeneration
More than 85% of the United States population requires repair or
replacement of a craniofacial structure, including bone, tooth,
temporomandibular joint, salivary gland, and mucosa. Regeneration of oral
and craniofacial tissues presents a formidable challenge that requires
synthesis of basic science, clinical science, and engineering technology.
Identification of appropriate scaffolds, cell sources, and spatial and temporal
signals are necessary to optimize development of a single tissue, hybrid
organs consisting of multiple tissues, or tissue interface. In contrast to
traditional replacement gene therapy, craniofacial regeneration via gene
therapy seeks to use genetic vectors as supplemental building blocks for
tissue growth and repair. Synergistic combination of viral gene therapy with
craniofacial tissue engineering will significantly enhance our ability to repair
and regenerate tissues in vivo.
CONCLUSION
Since the beginning of human gene therapy in 1990, nearly 1000 clinical trials
have been initiated. Patient follow-up for as much as 18 yrs post-gene transfer
has been generally positive, with isolated tragedy (Muul et al., 2003). It is
encouraging that many gene therapy trials for single-gene and comple
disorders are now complete, vector selection and design strategies have
significantly improved, and a safety profile is nearly established, as evidenced
by the many current phase III clinical trials. Though strategies such as ex
vivo transduction of cells with integrating retrovirus are promising, and early
success led to high hopes, it is essential to keep our expectations of gene
therapy realistic, because future development will require slow, stepwise
progress. As we near the 20-year mark for gene therapy and begin its integration
with craniofacial engineering, our focus must evolve to include expansion of
placebo-controlled clinical trials, development of targeted vectors to increase
transduction efficiency and to overcome the immune response, and
consideration of the concept of ‘genotoxicity’ testing as a fundamental feature
of gene therapy research.
https://www.ncbi.nlm.nih.gov

https://my.clevelandclinic.org

https://byjus.com

https://en.wikipedia.org

https://www.nature.com

https://medlineplus.gov