CELLULAR REGENERATION BASED ON DNA MODIFICATION

Medicina, Volume 27 - Edição 128/NOV 2023 / 28/11/2023

LA REGENERACIÓN CELULAR BASADA EN LA MODIFICACIÓN DEL ADN

REGISTRO DOI:10.5281/zenodo.10213769

Leandro de Oliveira Reckel1
Lucas de Brito Machado2
Filipe Flores Bicalho3
Pedro Flores Bicalho4
 Danylo Figueredo Cezana5
Dayra Fieni6
Aline Bonfante7
Bruno Pereira dos Santos8
Isabella Rocha Baggieri Santos9
Ana Lívia Ramalho Carretta10
Nayara Stefany Lacerda Brandão Fundeheller11

ABSTRACT

Genetic engineering has played a significant role in the field of medical education, serving as a specialized application in the diagnosis, treatment, and control of pathologies. Gene therapy is one of the methods employed by genetic engineering, involving the manipulation of genes using recombinant DNA. Two types of techniques are utilized: germline – the introduction of genetic material into spermatozoa or ova – and somatic – the introduction of genetic material into any other cells. One focus of this methodology is the correction of defective genes by introducing healthy genes into individuals with various disorders, thereby contributing to the genetic improvement of individuals.

Keywords: genetics, regeneration, modification, DNA, gene therapy.

RESUMEN

La ingeniería genética ha desempeñado un papel significativo en la ciencia de la educación médica al ser una especialidad aplicada en casos de diagnóstico, tratamiento y control de patologías. La terapia génica es uno de los métodos utilizados por la ingeniería genética, que implica la manipulación de los genes mediante el ADN recombinante. Hay dos tipos de técnicas utilizadas: la germinativa, que consiste en la introducción del material genético en los espermatozoides u ovocitos, y la somática, que implica la introducción del material genético en cualquier otra célula. Uno de los enfoques de esta metodología es la corrección de genes defectuosos mediante la introducción de genes saludables en portadores de diferentes trastornos, contribuyendo así a la mejora genética de los individuos.

Palabras clave: genética, regeneración, modificación, ADN, terapia génica.

             1.         CONCEPTS AND DEFINITION

Genetics and heredity, combined with molecular biology, constitute one of the outbreaks of new knowledge and technologies through which humanity is undergoing a conceptual renewal or recycling” (CONSOLARO et al., 2004). In this context, genetic engineering is characterized by a set of methodologies that enable the manipulation of         the      genome         of        living microorganisms,      with    the      consequent alteration        of        the      capabilities    of        each species,         according      to José Alberto Neves Candeias.

Within the universe related to genetics, some terms stand out as necessary for the understanding of this present work, such as:

Table 1 – Key Terms for Understanding the Article.

TERMOCONCEITO
Amino   acidSmall molecules invariably containing a carbon atom linked to the amine group, the hydroxyl group, and a hydrogen atom. There are 20 different types of amino acids. In a simplistic manner, it can be stated that a protein represents a sequence or chain of amino acids.
ProteinThey are macromolecules consisting of numerous amino acid molecules. Invariably, the amino acid has a carbon atom linked to an amino group, a hydroxyl group, and a hydrogen atom. When a protein has only a few amino acids, it can be called a peptide: di-, tri-, or polypeptide.
DNA“DNA or deoxyribonucleic acid: a macromolecule consisting of various nucleotides, each composed of a phosphate group, a deoxyribose sugar, and a nitrogenous base. The nitrogenous base can be Cytosine, Thymine, Adenine, or Guanine. Genetic information or memory is determined by the presence of these nitrogenous bases. The phosphate group and sugar serve purely structural functions in the molecule.”
RNAMacromolecule consisting of various nucleotides, each formed by a phosphate group, a ribose sugar, and a nitrogenous base. The nitrogenous base can be Cytosine, Uracil, Adenine, or Guanine. Thymine is exclusive to DNA, and Uracil to RNA.
GeneIt represents a sequence of nucleotides in DNA containing complete information or a complete set of instructions capable of guiding the cell to synthesize something or perform a specific function15. This segment of DNA, a sequence of nucleotides, or a file can vary in size; in other words, genes can be small or large. A gene may regulate a part of a structure, while another gene regulates another part of the same structure. In essence, genes can participate in the same process. Genes are sequentially located in DNA, or more precisely, they constitute DNA, and together with nuclear proteins, they form chromosomes. Each pair of chromosomes contains different genes. The specific location of a gene on the DNA molecule is also referred to as the gene
TERMOCONCEITO
 locus.

CONSOLARO et al, 2004 2. HISTORICAL CONTEXT OF GENETICS

Biotechnology, a set of techniques for organism selection, manipulation, and modification, emerges from the discovery of a compound called deoxyribonucleic acid (DNA), described as the key to the development and functioning of living beings (REIS, 2009). In 1869, biochemist Johann Friedrich Miescher revealed that proteins and lipids were the main components of cells, yet his analytical methods were simple protocols compared to the cellular protein diversity. During tests, Miescher found in the nucleus ‘a white, acidic substance rich in phosphorus,’ which he named ‘nuclein’ (MENCK, 2017).

According to Dahm (2004), Miescher confirmed that nuclein, unlike proteins, contained a large amount of phosphorus, along with elements commonly found in organic molecules, such as carbon, hydrogen, oxygen, and nitrogen. After experiments with leukocytes, the biochemist discovered the presence of nuclein in cells of other tissues, suggesting nuclei be classified based on nucleins’ presence, correlating with cell functions.

In 1910, Thomas Hunt Morgan demonstrated that chromosomes carry genes, inspiring the search for the chemical identification of the gene. The chemical arrangement of nucleic acids, formerly called nucleins, was later identified by the American Phoebus Aaron Levene, proposing a tetranucleotide structure for DNA (MENK, 2017). According to Moreira (2013), in 1928, Frederick Griffith observed that live virulent bacteria could transmit their virulence to non-pathogenic live bacteria. This information transmission became known as the ‘transforming principle,’ later explained in experiments describing DNA as the element responsible for genetic information transmission.

Figure 1 – Photograph of DNA molecules taken in 1950.

Source: Available at <https://www.quantumuniverse.nl/rosalind-franklin-en-haar-foto-vandna>.

Accessed on: October 26, 2023.

Francis Crick and James D. Watson concluded in 1951 that the DNA molecule is helical, and they also suggested that, in addition to the semi-conservative nature of DNA molecule duplication, ‘the transcription of the RNA molecule could be obtained by simple complementarity of one of the DNA strands’ (MENCK, 2017). In 1972, Paul Berg demonstrated the technology of recombinant DNA, based on a protocol for manipulating DNA from cells to produce specific proteins. Consequently, this technique allowed the creation of new gene combinations, often nonexistent in nature, through the location, isolation, alteration, and transplantation of DNA segments from one species to another (JACKSON, 1972).

In this context, it is evident that the expansion of knowledge about the human genome, coupled with advances in applied technology, enabled the direct manipulation of DNA through its recombination, according to the needs of the scientific community. Therefore, biotechnology and, consequently, genetic engineering began to be used as significant tools for genetic improvement (REIS, 2009).

3. EVOLUTION OF RESEARCH RELATED TO CROSSING AND GENE THERAPY

Gene therapy, through the development of techniques and subsequent protocols, has enabled the manipulation of an individual’s genetic makeup, despite encountering numerous challenges in certain procedures. From this perspective, this science holds the position of a “modern salvation” in addressing health-related issues, such as the cure of genetically inherited diseases or even conditions acquired during life, such as cancer, heart diseases, and viral infections (MENK, 2007).

Watson and Crick (1970) defined gene therapy as the ability to achieve genetic improvement by correcting altered (mutated) genes and/or specific codifications with the primary objective of therapeutic treatment. This procedure became feasible due to advances in genetic science and bioengineering, allowing the manipulation of vectors for the delivery of extrachromosomal material into target cells.

A lot of people claim to be concerned about the change in our genetic instructions. But these [genetic instructions] are merely a product of evolution, shaped to adapt us to certain conditions that may no longer exist. We all know how imperfect we are. Why not become a little more fit for survival?” (WATSON AND CRICK, 1970).

One of the most utilized techniques involves the application of recombinant DNA, where the gene of interest and/or a healthy gene is inserted into a vector, which can be plasmid, nanostructured, or viral. The latter is predominantly chosen for its efficiency in invading cells and introducing its genetic material. Despite various successful protocols, the gene therapy process remains complex, requiring ongoing developments (GONÇALVES, 2017).

Gonçalves (2017) further states that specific cells requiring treatment must be identified and arranged accessibly. From this perspective, an effective means of distributing gene copies to cells must be available, and both diseases and their close genetic connections need complete comprehension.

There is also a question regarding the target cell type in gene therapy, currently divided into two major groups: germline gene therapy and somatic cell gene therapy. In germline gene therapy, germ cells, such as sperm and egg cells, are modified by introducing functional genes that integrate into the genome. Changes are hereditary and pass on to subsequent generations. In theory, this approach should be highly effective in combating genetic and hereditary diseases (GONÇALVES, 2017).

According to the aforementioned author, somatic cell gene therapy involves transferring therapeutic genes to somatic cells of a patient. Any modification and effects are confined solely to the patient and are not inherited by future generations.

Additionally, there is the prevalence of a process known as RNA interference. In summary, this process involves a cellular mechanism responsible for post-transcriptional gene silencing (PTGS) acting on messenger RNA (mRNA). It relies on a double-stranded RNA (dsRNA) molecule that, when incorporated in its active form into an intracytoplasmic complex, binds to a complementary nucleotide sequence located on the target mRNA, thereby causing silencing through inhibition of translation and/or mRNA degradation (FRANÇA et al, 2010).

França et al (2010) asserts that there is evidence that dsRNAs are also involved in maintaining the condensation of chromatin regions and suppressing transcription in the vicinity of these regions. However, the association between transcriptional gene silencing (TGS) and RNAi is not yet fully clarified.

4. GENE THERAPY IN HEMOPHILIA B

Gene therapy has shown great success in some coagulopathies, notably hemophilia B, a recessive X-linked inherited disorder resulting from a deficiency in factor IX production in the liver, disrupting the coagulation mechanism. Symptoms typically include severe bleeding from minor injuries and even spontaneous hemorrhages. Additionally, there may be hematoma formation in muscles or joints, intracranial hemorrhage, or bleeding in vital organs (FLORES et al., 2004).

Flores et al. (2004) note that the initial diagnosis is made through blood coagulation tests, Activated Partial Thromboplastin Time (APTT), Coagulation Time (CT), Prothrombin Time (PT), Thrombin Time (TT), and Bleeding Time (BT). Once the diagnosis is confirmed, the treatment involves the application of a concentrated factor IX. Since coagulation factors are sourced from blood, there is a risk of contamination by agents such as HIV and hepatitis.

In cases like these, gene therapy proves to be of great utility, not only preventing the transmission of infectious agents but also yielding extremely positive results. In recent research led by Kay and colleagues in 1993, a virus, adeno-associated with gene 5 responsible for factor IX expression, was injected directly into the hepatic artery of severe hemophilia B patients. The vector caused no side effects, and the transgene expression achieved high therapeutic levels for eight weeks. It is also correct to state that in vivo therapy attains satisfactory levels of factor IX, even though the transgene may not express for an extended period (FARAH, 2007).”

         5. CURRENT       STATE      OF       HUMAN      DNA      MODIFICATION       AND

REGENERATION

The topic of ‘Human DNA Modification’ gained unprecedented attention in 2020 when scientists Emmanuelle Charpentier and Jennifer Doudna were awarded the 2020 Nobel Prize in Chemistry for their discovery of the groundbreaking genetic technology known as CRISPR/Cas9. This revolutionary gene-editing tool is contributing to new therapies for cancer and pioneering studies aimed at curing hereditary diseases.

The innovative features of the CRISPR/Cas9 system, particularly its ability to identify the system through base-pairing interactions between guide RNA and its target DNA, have positioned it ahead of other genetic therapy technologies (HEIDARI, 2015).

As stated by the aforementioned author, the meta-specificity of CRISPR/Cas9, coupled with its ease of use and effectiveness, has enabled its diverse applications in research, medicine, and biotechnology. Primarily designed as a genome-editing tool, CRISPR/Cas9 has been widely employed since 2013 across various cell lines and organisms, including mice, rats, fruit flies, nematodes, frogs, monkeys, as well as plants such as rice, wheat, soybeans, and tobacco. It has also been applied to fungi, organoids, embryonic stem cells, and induced pluripotent stem cells.

The CRISPR/Cas9 gene-editing tool functions as an associated protein, known as Cas. In summary, this CrisprCas9 technology allows direct targeting of defective genes as a guiding tool to reach specific target cells. In this process, the Cas9 enzyme (a type of nuclease) cuts both strands of double-stranded DNA, creating space for the insertion of new stretch sequences. This novel technology has transformed once-impossible genetic procedures into straightforward ‘cut and paste’ operations. It is essential to note that the primary purpose of utilizing this technology is to cure genetic diseases in the germline, affecting not only the patient but also their offspring and all subsequent generations. Although currently illegal, this technology paves the way for numerous new studies and opportunities in the field of genetic science (SGANZERLA, 2020).

According to Helinski (2013), in a genetic crossing study, the luciferase gene from the firefly Photinus pyralis was employed to produce light in plant cells and transgenic plants. In this process, a portion of the luciferase gene’s DNA was introduced into protoplast cells of plants (Daucus carota) through electroporation. As a result, the plants were able to produce light, and the luminescence pattern, though detected in most plant organs (leaves, stems, roots), seemed to reflect a specific organ distribution.”

Figure 2 – Photograph of a genetically modified leaf with the luciferase gene.

Source: Available at <https://www.sciencemag.org/>.

Accessed on: October 31, 2023.

The luciferase gene has been a crucial tool for studying gene expression in plant and animal cells. In genetic engineering, the luciferase gene proves to be uniquely valuable as a marker, paving the way for other procedures involving DNA modifications along with Genetic Crossing.

With the tremendous and rapid advancement in genetic engineering, human limb regeneration has garnered attention in the field of genetic science through cross breeding between species. Scientists conducted in-depth research on the axolotl, a salamander with remarkable regenerative capabilities. In 2018, its genome, ten times larger than the human genome, became the longest genome to be sequenced. However, understanding the function of genes involved in axolotl regeneration poses a challenge because these genes are contained in numerous repeated lengths of DNA.

Flores (2020) reports that researchers at Yale University have developed a new screening platform to bring the possibility of applying this regeneration process in humans closer. Increasingly, genetic technologies and resources have allowed researchers to explore the molecular origins of regenerative capacity in certain salamander species, such as axolotls, capable of fully regenerating limbs. Through various combinations, embryos, and general techniques, including CRISPR/Cas9 mutagenesis, genetic function, and the fidelity of limb regeneration can be explored. This enables the study of specific gene regeneration functions that may be necessary for other essential processes, such as organogenesis, morphological tissue changes, and other fundamental embryonic processes.

6. MATERIALS AND METHODS

In the development of this research, the following methodological steps were established: formulation of the guiding question, selection and acquisition of articles or books, evaluation of pre-selected studies, discussion of the obtained results, and the presentation of the integrative review.

The first step originated from the following guiding question: In what aspects can DNA modification promote the regeneration of cells, tissues, and limbs in humans?

The second step consisted of the selection of articles and books through searches in scientific literature, covering the period from July 1970 to April 2021, in the following databases: Virtual Health Library, Virtual Library of the University Center of Espírito Santo (UNESC), DataSUS, PubMed, and Springer.

The inclusion criteria established for the selection of scientific articles were: original articles that addressed the guiding question, with electronic availability in full text and published between 1970 and 2021, in Portuguese and/or English languages, and also containing, in their titles and/or abstracts, the following Health Science Descriptors (DeCS): ‘genetic therapy,’ ‘genetic therapy,’ ‘somatic gene therapy,’ and ‘gene therapy.’

Articles that did not meet the mentioned inclusion criteria were excluded.

7. INTEGRATIVE REVIEW AND DISCUSSION OF RESULTS

For the development of the theoretical framework of the study, 12 articles were used, following the selection criteria presented in the previous section. Among these articles, 6 were chosen to compose the integrative review and are presented in Table

2.”

Table 2 – Results and Conclusions of Selected Articles.

Authors/year            Article TitleResultsConclusions
Geraldo,     J.    M.,    & Montaño Valencia , C. J. 2023.Use  of  Nanostructured Systems    in     Cancer TreatmentNanomedicine, a convergent domain of nanotechnology and medicine, offers a variety of benefitsAmong the various lines of action that have been explored, genetic manipulation also emerges as a
  compared to the traditional therapeutic approach to cancer, such as increased sensitivity to radiation, versatility, effectiveness in drug administration, and regulated release of chemotherapeutic agents.promising suggestion to support cancer treatment, potentially in association with nanostructured systems to provide a synergistic effect. This could promote the inhibition of metastatic markers, increasing life expectancy in patients with more advanced stages.
   Sousa, et al. 2023.Osteogenic mesenchymal cells bone protein 9potential     of stem overexpressing morphogeneticBased on the literature analyzed, it is feasible to conclude that the amplification of CTM and BMP-9 enables the restoration of bone tissue in both in vivo and in vitro research. The procedure highlights the renewal process in extensive existing bone defects, being recognized as a promising and beneficial therapy in contrast to transplants.The application of tools enabling genomic analysis enhances the adaptability of the assessment context, providing expanded opportunities for bone formation in the field of health sciences compared to other regeneration approaches, such as currently existing autologous transplants.
  SOUZA, et al. 2015.  Regulation             and   Activation   of   Satellite   Cells   During   Muscle RegenerationIn the presence of muscle injury, quiescent satellite cells are activated by various growth and proliferation factors. Proliferation is positively influenced by HGF, IGF,The MGF, myogenin, and MRF4 stimulate the differentiation of myoblasts into myocytes, promoting muscle growth. Among the factors that negatively regulate the proliferation cycle of
  androgens,          ILinsulin. differentiation satellite          cells myoblasts stimulated myogenic MyoD, Myf5, VEGF.,  and The of into is by factors andSCs    are       non-steroidal anti-inflammatory drugs  and glucocorticoids, myostatin,      and          T3.
  Furtado, R. N. 2019.Genetic Editing: Risks and Benefits of Human   DNA ModificationGenetic modification techniques have been developed since the 1990s, representing, for some authors, a true revolution in the field    of biotechnology 4. The procedure is called this way because it is capable of ‘removing’ specific segments of DNA and introducing new genes in place—both germline and somatic cells can be modified 2. In the case of germline cells (eggs and sperm cells) and precursor cells, genetic alterations are passed on to descendants. Some researchers also include embryos in the early stage of formation under this designation. On the other hand, somatic cells refer to all otherThe editing occurs in two steps: first, identification and cutting of the DNA, then, repair of the molecule. Four techniques are used: 1) meganucleases; 2) zinc-finger nucleases; 3) transcription activator-like effector nucleases; and 4) CRISPR-Cas9. These tools have recognition devices to adhere to specific sequences of the target DNA and cleavage devices to cut the nucleotides. After the cut, double-strand breaks occur, triggering cellular repair mechanisms, such as NHEJ to silence genes and HDR to regenerate double-strand breaks using          external          DNA templates.
  cells in the organism, but their changes are not hereditary. 
Oliveira, C. S., Nascimento, M., de Almeida Junior, E., Crusoé, M., Bahia, P., & Rosa, F. P. 2010.Advances              and Applications  of  Tissue BioengineeringCurrently, there are still many controversies regarding the ethical, political, and social issues related to the use of stem cells for therapeutic applications.            Despite this,      several investigations have already been conducted with various types of existing stem cells, as discussed. However,             more comprehensive molecular-level studies are essential to precisely understand the factors influencing stem     cell differentiation.The combination of gene therapy, tissue engineering, and biomaterials has the potential to create synthetic environments that provide the necessary stimuli to promote the formation of functional tissues. Significant progress has been made in utilizing structures that facilitate the delivery of cells, genes, and proteins to injuries. For new biomaterials to be successfully employed in tissue engineering, there is an essential need to investigate the interactions between cells and these materials.
Martins,        R.       S., Siqueira, M. G., Silva, C. F., Plese, J. P. P. 2005.Basic   Mechanisms  of Nerve RegenerationIf axon reconstruction does not occur, changes may arise in the target organs. Muscle fibers atrophy, assuming a more rounded shape from the originally peripheral to central part of the cell. Some of these changes canIt is noted that the renewal is an intricate mechanism whose effectiveness depends on the occurrence and integration of various phases subject to numerous cellular and molecular signals. Thus, it is easy to understand that
  be observed a few weeks after the injury. Neuromuscular junctions also shrink and disappear, a process that begins three months after axonal injury. Muscle tissue is replaced by fibrotic tissue between 12 and 24 months after nerve injury.although the renewal process is highly effective when observed in its entirety, the absence of a step results in the detriment of the entire process, leading to unsatisfactory clinical consequences. The challenge for the coming decades is to translate, for clinical application, all the advances achieved from understanding renewal, enhancing success rates after surgical treatment of traumatic nerve injuries.
8. CONCLUSION

Understanding and manipulating the genetic code have paved revolutionary paths in science and medicine. The fundamental molecule, DNA, serves as the cornerstone of genetic information, with the nucleotide sequence determining cellular characteristics and functions. The historical evolution, from Miescher’s discoveries to Watson and Crick’s revelation of DNA’s helical structure, has shaped our understanding of heredity. Biotecnology has emerged from this knowledge, and gene therapy has become a ‘modern salvation’ for various genetic and acquired conditions. The DNA recombinant technique, introduced by Paul Berg, has enabled unprecedented genetic manipulations, creating new gene combinations and, consequently, advancing therapeutic possibilities. Hemophilia B exemplifies the success of gene therapy, where introducing the factor IX gene through viral vectors has yielded positive results, avoiding risks associated with conventional therapy. Moreover, the CRISPR/Cas9 technology has revolutionized genetic editing, providing a precise and efficient tool for correcting defective genes. The ability to modify human DNA, exemplified by the 2020 Nobel Prize in Chemistry, marks a milestone in genetic science.

The CRISPR/Cas9 genetic scissors, with their target specificity, have opened new experimental opportunities, allowing precise gene editing in various species. Human DNA regeneration and modification, notably the regenerative capacity of the axolotl, point toward a promising future. Genetic research, coupled with genetic crossing and CRISPR/Cas9 technology, provides insights into human limb regeneration. Understanding the genes involved in axolotl regeneration propels research into specific functions, opening doors to innovative therapeutic applications. In summary, the modification of human DNA is a fascinating and promising journey with profound implications for medicine and our understanding of life.

As we push the boundaries of genetic engineering, new therapeutic possibilities and scientific advancements emerge, pointing toward a future where genetic manipulation may become a common tool in the pursuit of health and disease cure.

9. REFERENCES
  1. CANDEIAS, José Alberto Neves. A engenharia genética. *Revista de Saúde Pública*, São Paulo, v.1, n. 25, fev. 1991.
  2. CONSOLARO, Alberto, et al. Conceitos de genética e hereditariedade aplicados à compreensão das reabsorções dentárias durante a movimentação ortodôntica. *Revista Dental Press de Ortodontia e Ortopedia Facial*, Maringá, v. 9, n. 2, p. 79-94, abr. 2004.
  3. DAHM, Ralf. Friedrich Miescher and the discovery of DNA. *Revista Elsevier*, Amsterdam, n. 2, fev. 2005.
  4. FARAH, Solange Bento. *DNA: segredos e mistérios*. 2. ed. São Paulo: Sarvier, 2007.
  5. FLORES, Rafael Py Gonçalves et al. Hemofilia e anestesia. *Revista Brasileira de Anestesiologia*, Campinas, v. 54, n. 6, p. 865-871, 2004.
  6. FRANÇA, N. R. de et al. Interferência por RNA: Uma nova alternativa para terapia nas doenças reumáticas. *Revista Brasileira de Reumatologia*, São Paulo, v. 50, n. 10, p. 695-709, 2010.
  7. GONÇALVES, Giulliana Augusta Rangel; PAIVA, Raquel de Melo Alves. Terapia gênica: avanços, desafios e perspectivas. *Revista Einstein*, São Paulo, 2017.
  8. JACKSON, David A.; SYMONS, Robert H.; BERG, Paul. Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. *Stanford University Medical Center*, Stanford, v. 69, n. 10, jul. 1972.
  9. MENCK, Carlos F. M.; SLUYS, Marie-Anne Van. *Genética Molecular Básica: dos genes aos genomas*. 1. ed. Rio de Janeiro: Guanabara Koogan, p. 528, 2017.
  10. MENCK, Carlos F. M.; VENTURA, Armando M. Manipulando genes em busca de cura: o futuro da terapia gênica. *Revista USP*, São Paulo, n. 75, p. 50-61, 2007.
  11. MOREIRA, Catarina. *DNA*. *Revista de Ciência Elementar*, Lisboa, v. 1, n. 1, dez. 2013.
  12. PIMENTA, Célia Aparecida Marques; LIMA, Jacqueline Miranda de. *Genética Aplicada à Biotecnologia*. 1. ed. São Paulo: Érica, 2015. 112p.
  13. REIS, Carla et al. Biotecnologia para saúde humana: tecnologias, aplicações e inserção na indústria farmacêutica. *BNDES Setorial*, Rio de Janeiro, n. 29, 2009. 14. Geraldo, J. M., & Montaño Valencia , C. J. (2023). Uso de sistemas nanoestruturados no tratamento do câncer: da radioterapia à terapia genética. *Mário Penna Journal*, 1(1), 18–33. https://doi.org/10.61229/mpj.v1i1.4
  14. SOUSA, Z. da S. .; MOREIRA, T. P. C. .; CHAVES JÚNIOR, P. M. .; MATIAS, A. C. da S. .; SANTOS, V. P. dos .; MATEUS, M. M. .; MARQUES, D. L. .; SILVA, S. E. da; ALVES, J. R. de S.; SÁ, B. D. .; ANDRADE, M. C. . Potencial osteogênico de células-tronco mesenquimais sobre-expressão da proteína morfogenética óssea 9. *Revista de Casos e Consultoria*, [S. l.], v. 14, n. 1, p. e30285, 2023.
  15. SOUZA, DK; OLIVEIRA, JR; RODRIGUES, H; COTA, NB; CARVALHO, MM; PRESTES, J; DURIGAN, JLQ; PEREIRA, ECL. Regulação e ativação das células satélites durante a regeneração muscular. *R. bras. Ci. e Mov* 2015;23(3):170-180. 17. FURTADO, Rafael Nogueira. Edição genética: riscos e benefícios da modificação do DNA humano. *Rev. Bioét.*, vol. 27, no. 2, Brasília, Abr./Jun. 2019.
  16. Oliveira, C. S., Nascimento, M., de Almeida Junior, E., Crusoé, M., Bahia, P., &

Rosa, F. P. (2010). Avanços e aplicações da bioengenharia tecidual. *Revista De

Ciências        Médicas         E         Biológicas*,  9(1),    28–36. https://doi.org/10.9771/cmbio.v9i1.4729 MARTINS, R. S., SIQUEIRA, M. G., SILVA, C. F., PLESE, J. P. P. Mecanismos básicos da regeneração de nervos. *Arq Bras Neurocir* 24(1

Leandro de Oliveira Reckel- Graduando em Medicina Centro Universitário do Espírito Santo – UNESC Avenida Fioravante Rossi, 2930 – Bairro Martinelli – Colatina/ES CEP 29703-8581
Lucas de Brito Machado – Graduando em Medicina Centro Universitário do Espírito Santo – UNESC Avenida Fioravante Rossi, 2930 – Bairro Martinelli – Colatina/ES CEP 29703-8582
Filipe Flores Bicalho – Graduando em Medicina Centro Universitário do Espírito Santo – UNESC Avenida Fioravante Rossi, 2930 – Bairro Martinelli – Colatina/ES CEP 29703-8583
Pedro Flores Bicalho – Graduando em Medicina Centro Universitário do Espírito Santo – UNESCAvenida Fioravante Rossi, 2930 – Bairro Martinelli – Colatina/ES CEP 29703-8584
Danylo Figueredo Cezana – Graduando em Medicina Centro Universitário do Espírito Santo – UNESC Avenida Fioravante Rossi, 2930 – Bairro Martinelli – Colatina/ES CEP 29703-855
Dayra Fieni – Graduando em Medicina Centro Universitário do Espírito Santo – UNESC
Avenida Fioravante Rossi, 2930 – Bairro Martinelli – Colatina/ES CEP 29703-8586
Aline Bonfante – Graduando em Medicina Centro Universitário do Espírito Santo – UNESC Avenida Fioravante Rossi, 2930 – Bairro Martinelli – Colatina/ES CEP 29703-8587
Bruno Pereira dos Santos – Graduando em Medicina Centro Universitário do Espírito Santo – UNESC
Avenida Fioravante Rossi, 2930 – Bairro Martinelli – Colatina/ES CEP 29703-858
Isabella Rocha Baggieri Santos – Pós Graduanda em Medicina da Família e Comunidade Universidade Federal de Santa Catarina R. Eng. Agronômico Andrei Cristian Ferreira, s/n – Trindade, Florianópolis – SC, CEP 88040-9009
Ana Lívia Ramalho Carretta – Graduando em Medicina Centro Universitário do Espírito Santo – UNESC Avenida Fioravante Rossi, 2930 – Bairro Martinelli – Colatina/ES CEP 29703-85810
Nayara Stefany Lacerda Brandão Fundeheller – Graduando em Medicina Centro Universitário do Espírito Santo – UNESC Avenida Fioravante Rossi, 2930 – Bairro Martinelli – Colatina/ES11