Slow and steady wins the race: Fractionated near-infrared treatment empowered by graphene-enhanced 3D scaffolds for precision oncology.

Surgically addressing tumors poses a challenge, requiring a tailored, multidisciplinary approach for each patient based on the unique aspects of their case. Innovative therapeutic regimens combined to reliable reconstructive methods can contribute to an extended patient's life expectancy. This study presents a detailed comparative investigation of near-infrared therapy protocols, examining the impact of non-fractionated and fractionated irradiation regimens on cancer treatment. The therapy is based on the implantation of graphene oxide/poly(lactic-co-glycolic acid) three-dimensional printed scaffolds, exploring their versatile applications in oncology by the examination of pro-inflammatory cytokine secretion, immune response, and in vitro and in vivo tumor therapy. The investigation into cell death patterns (apoptosis vs necrosis) underlines the pivotal role of protocol selection underscores the critical influence of treatment duration on cell fate, establishing a crucial parameter in therapeutic decision-making. In vivo experiments corroborated the profound impact of protocol selection on tumor response. The fractionated regimen emerged as the standout performer, achieving a substantial reduction in tumor size over time, surpassing the efficacy of the non-fractionated approach. Additionally, the fractionated regimen exhibited efficacy also in targeting tumors in proximity but not in direct contact to the scaffolds. Our results address a critical gap in current research, highlighting the absence of a standardized protocol for optimizing the outcome of photodynamic therapy. The findings underscore the importance of personalized treatment strategies in achieving optimal therapeutic efficacy for precision cancer therapy.

Severely Damaged Freeze-Injured Skeletal Muscle Reveals Functional Impairment, Inadequate Repair, and Opportunity for Human Stem Cell Application.

BACKGROUND: The regeneration of severe traumatic muscle injuries is an unsolved medical need that is relevant for civilian and military medicine. In this work, we produced a critically sized nonhealing muscle defect in a mouse model to investigate muscle degeneration/healing phases. MATERIALS AND METHODS: We caused a freeze injury (FI) in the biceps femoris of C57BL/6N mice. From day 1 to day 25 post-injury, we conducted histological/morphometric examinations, an analysis of the expression of genes involved in inflammation/regeneration, and an in vivo functional evaluation. RESULTS: We found that FI activates cytosolic DNA sensing and inflammatory responses. Persistent macrophage infiltration, the prolonged expression of eMHC, the presence of centrally nucleated myofibers, and the presence of PAX7+ satellite cells at late time points and with chronic physical impairment indicated inadequate repair. By looking at stem-cell-based therapeutic protocols of muscle repair, we investigated the crosstalk between M1-biased macrophages and human amniotic mesenchymal stem cells (hAMSCs) in vitro. We demonstrated their reciprocal paracrine effects where hAMSCs induced a shift of M1 macrophages into an anti-inflammatory phenotype, and M1 macrophages promoted an increase in the expression of hAMSC immunomodulatory factors. CONCLUSIONS: Our findings support the rationale for the future use of our injury model to exploit the full potential of in vivo hAMSC transplantation following severe traumatic injuries.

DNA vaccination strategies for anti-tumour effective gene therapy protocols.

After more than 15 years of experimentation, DNA vaccines have become a promising perspective for tumour diseases, and animal models are widely used to study the biological features of human cancer progression and to test the efficacy of vaccination protocols. In recent years, immunisation with naked plasmid DNA encoding tumour-associated antigens or tumour-specific antigens has revealed a number of advantages: antigen-specific DNA vaccination stimulates both cellular and humoral immune responses; multiple or multi-gene vectors encoding several antigens/determinants and immune-modulatory molecules can be delivered as single administration; DNA vaccination does not induce autoimmune disease in normal animals; DNA vaccines based on plasmid vectors can be produced and tested rapidly and economically. However, DNA vaccines have shown low immunogenicity when tested in human clinical trials, and compared with traditional vaccines, they induce weak immune responses. Therefore, the improvement of vaccine efficacy has become a critical goal in the development of effective DNA vaccination protocols for anti-tumour therapy. Several strategies are taken into account for improving the DNA vaccination efficacy, such as antigen optimisation, use of adjuvants and delivery systems like electroporation, co-expression of cytokines and co-stimulatory molecules in the same vector, different vaccination protocols. In this review we discuss how the combination of these approaches may contribute to the development of more effective DNA vaccination protocols for the therapy of lymphoma in a mouse model.

DNA vaccines: developing new strategies against cancer.

Due to their rapid and widespread development, DNA vaccines have entered into a variety of human clinical trials for vaccines against various diseases including cancer. Evidence that DNA vaccines are well tolerated and have an excellent safety profile proved to be of advantage as many clinical trials combines the first phase with the second, saving both time and money. It is clear from the results obtained in clinical trials that such DNA vaccines require much improvement in antigen expression and delivery methods to make them sufficiently effective in the clinic. Similarly, it is clear that additional strategies are required to activate effective immunity against poorly immunogenic tumor antigens. Engineering vaccine design for manipulating antigen presentation and processing pathways is one of the most important aspects that can be easily handled in the DNA vaccine technology. Several approaches have been investigated including DNA vaccine engineering, co-delivery of immunomodulatory molecules, safe routes of administration, prime-boost regimen and strategies to break the immunosuppressive networks mechanisms adopted by malignant cells to prevent immune cell function. Combined or single strategies to enhance the efficacy and immunogenicity of DNA vaccines are applied in completed and ongoing clinical trials, where the safety and tolerability of the DNA platform are substantiated. In this review on DNA vaccines, salient aspects on this topic going from basic research to the clinic are evaluated. Some representative DNA cancer vaccine studies are also discussed.

Genetic immunization with CDR3-based fusion vaccine confers protection and long-term tumor-free survival in a mouse model of lymphoma.

Therapeutic vaccination against idiotype is a promising strategy for immunotherapy of B-cell malignancies. We have previously shown that CDR3-based DNA immunization can induce immune response against lymphoma and explored this strategy to provide protection in a murine B-cell lymphoma model. Here we performed vaccination employing as immunogen a naked DNA fusion product. The DNA vaccine was generated following fusion of a sequence derived from tetanus toxin fragment C to the V(H)CDR3(109-116) epitope. Induction of tumor-specific immunity as well as ability to inhibit growth of the aggressive 38C13 lymphoma and to prolong survival of vaccinated mice has been tested. We determined that DNA fusion vaccine induced immune response, elicited a strong protective antitumor immunity, and ensured almost complete long-term tumor-free survival of vaccinated mice. Our results show that CDR3-based DNA fusion vaccines hold promise for vaccination against lymphoma.

The Innate Immune Signalling Pathways: Turning RIG-I Sensor Activation Against Cancer.

Over the last 15 years, the ability to harness a patient's own immune system has led to significant progress in cancer therapy. For instance, immunotherapeutic strategies, including checkpoint inhibitors or adoptive cell therapy using chimeric antigen receptor T-cell (CAR-T), are specifically aimed at enhancing adaptive anti-tumour immunity. Several research groups demonstrated that adaptive anti-tumour immunity is highly sustained by innate immune responses. Host innate immunity provides the first line of defence and mediates recognition of danger signals through pattern recognition receptors (PRRs), such as cytosolic sensors of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular pattern (DAMP) signals. The retinoic acid-inducible gene I (RIG-I) is a cytosolic RNA helicase, which detects viral double-strand RNA and, once activated, triggers signalling pathways, converging on the production of type I interferons, proinflammatory cytokines, and programmed cell death. Approaches aimed at activating RIG-I within cancers are being explored as novel therapeutic treatments to generate an inflammatory tumour microenvironment and to facilitate cytotoxic T-cell cross-priming and infiltration. Here, we provide an overview of studies regarding the role of RIG-I signalling in the tumour microenvironment, and the most recent preclinical studies that employ RIG-I agonists. Lastly, we present a selection of clinical trials designed to prove the antitumour role of RIG I and that may result in improved therapeutic outcomes for cancer patients.

Anti-tumor immunity induced by CDR3-based DNA vaccination in a murine B-cell lymphoma model.

The idiotypic structure present on B-cell neoplasms is a tumor-specific antigen and an attractive target for immunotherapy. Here, the tumor protective effects recruited by CDR3-based DNA vaccines in the poorly immunogenic, highly aggressive 38C13 murine B-cell lymphoma model were evaluated. The regions belonging to the idiotypic V(H) and V(L) CDR3 sequences were chosen for the design of two synthetic mini-genes and arranged in high-level expression plasmids. Syngeneic C3H/HeN mice were immunized by intramuscular electroporation with pV(H)CDR3-IL-2 and pV(L)CDR3-IL-2 naked DNAs. This approach provided protection in about 60% of animals challenged with a 2-fold lethal dose of tumor cells, as opposed to non-survivors in control groups. Furthermore, a long-term survival was induced in these mice since they were still alive and tumor-free 4 months following tumor challenge. Analysis of the humoral immunity revealed the presence of antibodies reactive with the peptides encompassing the CDR3 sequences in the sera of vaccinated mice. Moreover, immune sera specifically reacted with the parental 38C13 tumor cells in flow cytometry assays, indicating that such immunization elicited anti-idiotypic antibodies. These findings provide a basis for exploring the use of CDR3-based DNA vaccines against B-cell lymphoma.

Nucleic Acid Sensing Machinery: Targeting Innate Immune System for Cancer Therapy.

BACKGROUND: Nucleic acid sensing is an essential strategy employed by the innate immune system to detect both pathogen-derived nucleic acids and self-DNA released by host apoptotic or necrotic cells. The presence of nucleic acids that gain access to the cytoplasm is perceived by mammalian cells as "stranger" or "danger" signals that trigger a myriad of immunological responses. Recent publications have highlighted the importance of nucleic acid sensing machinery as mediator of innate and adaptive immunity, and cGAS, STING and RIG-I agonists have been validated as immunooncology agents in cancer therapy. OBJECTIVE: The crucial role of cGAS and STING in eliciting innate and adaptive immune responses provides a scientific rationale for using cGAMP and STING agonists both in human preventive vaccine and immunotherapy settings. Thus, search for natural and synthetic STING agonists and development of cyclic dinucleotides (CDNs)-based adjuvants were strongly intensified. Furthermore, with their ability to induce tumour cell death and lymphocyte cross priming, RIG-I ligands are among the most promising molecules for the development of new immunostimulatory adjuvants in cancer vaccines. RESULTS: This work focuses on relevant recent patents (2010-2017) that entail the use of nucleic acid sensing machinery to elicit innate and adaptive immune responses, highlighting a new approach in immune-mediated cancer therapy. Several patents describe compositions and methods that may be used as immuno-oncology agents for the treatment of cancer patients. cGAS and/or STING pathways modulating compounds alone or in combination with pharmaceutical compositions are discussed. New approaches to improve DNA-vaccine induced adaptive immunity for cancer therapy through increasing the level of plasmid-mediated activation of innate immune signalling pathways are also discussed. In addition, a targeted selection of very recent clinical studies describing the employment of innate immunity targeting compounds is reported. CONCLUSION: It is highly relevant to deepen the study of the nucleic acid-sensing mechanisms to develop new pharmacological approaches to engage these pathways within the tumour microenvironment. Indeed, further clarification will be functional to develop advanced anticancer strategies or to design new vaccine formulations.

Targeting Cytosolic Nucleic Acid-Sensing Pathways for Cancer Immunotherapies.

The innate immune system provides the first line of defense against pathogen infection though also influences pathways involved in cancer immunosurveillance. The innate immune system relies on a limited set of germ line-encoded sensors termed pattern recognition receptors (PRRs), signaling proteins and immune response factors. Cytosolic receptors mediate recognition of danger damage-associated molecular patterns (DAMPs) signals. Once activated, these sensors trigger multiple signaling cascades, converging on the production of type I interferons and proinflammatory cytokines. Recent studies revealed that PRRs respond to nucleic acids (NA) released by dying, damaged, cancer cells, as danger DAMPs signals, and presence of signaling proteins across cancer types suggests that these signaling mechanisms may be involved in cancer biology. DAMPs play important roles in shaping adaptive immune responses through the activation of innate immune cells and immunological response to danger DAMPs signals is crucial for the host response to cancer and tumor rejection. Furthermore, PRRs mediate the response to NA in several vaccination strategies, including DNA immunization. As route of double-strand DNA intracellular entry, DNA immunization leads to expression of key components of cytosolic NA-sensing pathways. The involvement of NA-sensing mechanisms in the antitumor response makes these pathways attractive drug targets. Natural and synthetic agonists of NA-sensing pathways can trigger cell death in malignant cells, recruit immune cells, such as DCs, CD8+ T cells, and NK cells, into the tumor microenvironment and are being explored as promising adjuvants in cancer immunotherapies. In this minireview, we discuss how cGAS-STING and RIG-I-MAVS pathways have been targeted for cancer treatment in preclinical translational researches. In addition, we present a targeted selection of recent clinical trials employing agonists of cytosolic NA-sensing pathways showing how these pathways are currently being targeted for clinical application in oncology.

Looking Beyond the 5-HTTLPR Polymorphism: Genetic and Epigenetic Layers of Regulation Affecting the Serotonin Transporter Gene Expression.

Serotonin (5-HT) is a neurotransmitter that regulates fundamental aspects of brain development, physiology and behaviour. The serotonin transporter (5-HTT) is deputized to the reuptake of 5-HT from the intersynaptic space in the presynaptic neurons. 5-HTT governs duration and magnitude of 5-HT biological actions, acting as a master regulator of the fine-tuning of 5-HT signalling. Genetic variation at SLC6A4 gene locus, encoding 5-HTT, contributes to alteration in 5-HT reuptake. The 5-HTTLPR/rs25531/rs25532 polymorphisms located in the promoter region of SLC6A4 gene have been associated with stress-related psychopathology and functional brain phenotypes. Besides, further DNA variations in functional regulative elements located at 5' and 3' termini of the SLC6A4 gene influence transcriptional and post-transcriptional steps. Recently, epigenetic processes including SLC6A4 promoter methylation and transcript silencing by microRNA were shown to be involved in the aetiology of affective disorders. Furthermore, gene-environment interactions such as early life stress often encompass epigenetic changes, which can stably mark the genome in response to environmental stimuli potentially altering gene expression across lifespan. Therefore, it seems well established that functional variations in the SLC6A4 gene expression can no longer be ascribed to the modulating 5-HTTLPR promoter polymorphism but need to be integrated with the contribution arising from other interactive elements and epigenetic mechanisms. In this review, we discuss genetic and epigenetic layers of regulation affecting SLC6A4 gene expression. An overview of human and cellular studies investigating the impact of these regulatory processes on SLC6A4 gene expression is provided.

Role of the 5-HTTLPR and SNP Promoter Polymorphisms on Serotonin Transporter Gene Expression: a Closer Look at Genetic Architecture and In Vitro Functional Studies of Common and Uncommon Allelic Variants.

The serotonin (5-hydroxytriptamine (5-HT)) transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) is a variable number tandem repeats (VNTR) located in the promoter region of the human 5-HTT-encoding gene SLC6A4. This length polymorphism gives rise to different promoter variants, variously influencing SLC6A4 expression. Over the years, an extensive literature has investigated the relationships between these promoter variants and SLC6A4 gene expression, since these variants have been variously associated to complex neuropsychiatric conditions and traits. In this review, we detail the genetic architecture of the 5-HTTLPR allelic variants reported so far, with a closer look at the two single nucleotide polymorphisms (SNPs) rs25531 and rs25532 that lies in the VNTR and thus increase genetic variability of the SLC6A4 promoter. We summarize the hypothesized molecular mechanisms underlying this variation. We also provide an update on common and uncommon 5-HTTLPR allelic variants reviewing the available data on functional in vitro analysis of their regulatory effect on SLC6A4 gene transcription. Controversial findings are highlighted and critically discussed. A deeper knowledge of the "5-HTTLPR universe" will be useful to better understand the molecular basis of serotonin homeostasis and the pathological basis underlying serotonin-related neuropsychiatric conditions and traits.

Recent advances in design of immunogenic and effective naked DNA vaccines against cancer.

A variety of clinical trials for vaccines against cancer have provided evidence that DNA vaccines are well tolerated and have an excellent safety profile. DNA vaccines require much improvement to make them sufficiently effective against cancer in the clinic. Nowadays, it is clear that an increased antigen expression correlates with improved immunogenicity and it is critical to vaccine performance in large animals and humans. Similarly, additional strategies are required to activate effective immunity against poorly immunogenic tumour antigens. This review discusses very recent scientific references focused on the development of sophisticated DNA vaccines against cancer. We report a selection of novel and relevant patents employed to improve their immunogenicity through several strategies such as the use of tissue-specific transcriptional elements, nuclear localisation signalling, codon-optimisation and by targeting antigenic proteins to secretory pathway. Recent patents validating portions or splice variants of tumour antigens as candidates for cancer DNA vaccines with improved specificity, such as mesothelin and hTERT, are also discussed. Lastly, we review novel patents on the use of genetic immunomodulators, such as "universal" T helper epitopes derived from tetanus toxin, E. coli heat labile enterotoxin and vegetable proteins, as well as cytokines, chemokines or costimulatory molecules such as IL-6, IL-15, IL- 21 to amplify immunity against cancer.

A blueprint for DNA vaccine design.

Although safety concerns have been overcome, lower immunogenicity profiles of DNA vaccines have hindered their progress in humans. DNA vaccines need to make up for this limitation by altering plasmid construction through vector design innovations intended for enhancement of transgene expression and immunogenicity. The next-generation vectors also address safety issues such as selection markers. This chapter discusses (a) plasmid backbone design, (b) enhancement of antigenic protein expression and immunogenicity, and (c) vector modification to increase innate immunity. Modifications of the basic design, when combined with improved delivery devices and/or prime/boost regimens, may enhance DNA vaccine performance and clinical outcomes.

Enhancement of plasmid-mediated transgene expression.

A large number of studies aimed at the treatment of cancer, autoimmune and metabolic diseases, neurodegenerative disorders, allergic diseases, as well as muscle disorders strengthen the fact that gene therapy could represent an alternative method to treat human diseases where conventional approaches are less effective. To improve transgene expression from plasmid vectors, DNA nuclear targeting sequences (DTSs) can be introduced in a vector backbone to increase in vivo expression up to 20-fold using electroporation (EP) delivery in muscle tissue. The purpose of this chapter is to represent a step-by-step strategy for the construction of a plasmid vector with enhanced efficiency of nuclear plasmid uptake and the methodic for the in vivo efficiency evaluation of the obtained expression vector.

Strategies for improving DNA vaccine performance.

The goal of active vaccination is to induce all the immune effector pathways and to establish immunological memory allowing prolonged surveillance against pathogens or cancer cells. DNA vaccination platform is an intriguing strategy owing to its ability to mobilize both branches of the immune system (i.e., innate immunity as well as adaptive immunity). Since plasmids offer several advantages for biotechnological applications due to their modular structure and easy manipulation, a wide range of strategies can be applied to improve DNA vaccine performance. This chapter discusses this topic in detail taking into account antigen/epitope selection and optimization, inclusion of intracellular targeting sequences and genetic adjuvants, and provision of T cell help.

In vivo DNA electrotransfer for immunotherapy of cancer and neurodegenerative diseases.

Electroporation is the process commonly referred to the transient increase in the permeability of cell membranes on submission to electric field pulses. Electroporation has become an increasingly extensive method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery of DNA vaccines, mostly against cancer. In vivo gene electrotransfer is of special interest since it is the most efficient non-viral strategy for gene delivery and it is worthy of low manufacturing costs, ease of realization and favorable safety profile. No adverse findings observed in toxicology and biodistribution/integration studies have been warranted for the evaluation of this approach in humans. Therefore, gene delivery followed by electroporation is currently being investigated in several clinical trials. The positive outcomes of early studies suggest that the efficacy of gene delivery and immunogenicity has greatly improved by electroporation. This review briefly summarizes salient features and recent findings that have contributed to the rapid progress of electroimmunotherapy as well as an overview of advanced clinical studies in oncology. Translation of in vivo DNA electrovaccination for neurodegenerative diseases as well as future expectations are also discussed.

Epitope-driven DNA vaccine design employing immunoinformatics against B-cell lymphoma: a biotech's challenge.

DNA vaccination has been widely explored to develop new, alternative and efficient vaccines for cancer immunotherapy. DNA vaccines offer several benefits such as specific targeting, use of multiple genes to enhance immunity and reduced risk compared to conventional vaccines. Rapid developments in molecular biology and immunoinformatics enable rational design approaches. These technologies allow construction of DNA vaccines encoding selected tumor antigens together with molecules to direct and amplify the desired effector pathways, as well as highly targeted vaccines aimed at specific epitopes. Reliable predictions of immunogenic T cell epitope peptides are crucial for rational vaccine design and represent a key problem in immunoinformatics. Computational approaches have been developed to facilitate the process of epitope detection and show potential applications to the immunotherapeutic treatment of cancer. In this review a number of different epitope prediction methods are briefly illustrated and effective use of these resources to support experimental studies is described. Epitope-driven vaccine design employs these bioinformatics algorithms to identify potential targets of vaccines against cancer. In this paper the selection of T cell epitopes to develop epitope-based vaccines, the need for CD4(+) T cell help for improved vaccines and the assessment of vaccine performance against tumor are reviewed. We focused on two applications, namely prediction of novel T cell epitopes and epitope enhancement by sequence modification, and combined rationale design with bioinformatics for creation of new synthetic mini-genes. This review describes the development of epitope-based DNA vaccines and their antitumor effects in preclinical research against B-cell lymphoma, corroborating the usefulness of this platform as a potential tool for cancer therapy. Achievements in the field of DNA vaccines allow to overcome hurdles to clinical translation. In a scenario where the vaccine industry is rapidly changing from a mostly empirical approach to a rational design approach, these new technologies promise to discover and develop high-value vaccines, creating a new opportunity for future markets.

Design and pre-clinical development of epitope-based DNA vaccines against B-cell lymphoma.

Optimally designed cancer vaccines should combine the best tumor antigens with the most effective immunotherapy agents and delivery strategies to achieve positive clinical results. The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a cancer vaccine. It has been theorized that effective cancer vaccines can be developed using the minimum essential subset of T cell and B cell epitopes that comprise the 'immunome', the universe of neoplasm-derived peptides that interface with B and T cells of the host immune system. Idiotypic antigenic determinants of a B-cell lymphoma lie within the hypervariable regions and mainly within the complementarity-determining regions (CDR)s 3. Thus, the CDR3s are considered a "hot spot" of particular interest for construction of subunit vaccines. DNA vaccines, whose safety and tolerability are substantiated in completed and ongoing clinical trials, have emerged as a novel lymphoma vaccine formulation for antigen-specific immunotherapy. The molecular precision tools offered by gene-based vaccines allow to explore the use of CDR3 sequence as an anti-lymphoma vaccine.

The pathological cross talk between apolipoprotein E and amyloid-beta peptide in Alzheimer's disease: emerging gene-based therapeutic approaches.

Apolipoprotein E (ApoE) plays a key role in lipid transport in the plasma and in the central nervous system through its interaction with members of the low-density lipoprotein receptor family. The three common isoforms of ApoE (ApoE2, ApoE3, and ApoE4) differ in their ability to perform neuronal maintenance and repair functions and differentially affect the risk of developing neurodegenerative disorders. The ApoE4 isoform is a strong genetic risk factor for Alzheimer's disease. Up-to-date knowledge about the structural and biophysical features of ApoE4 shed light on the molecular basis underlying the isoform-specific pathogenic role of ApoE4 and its contribution to AD pathology through several different mechanisms. ApoE4 impacts on amyloid-beta (Abeta) production, Abeta clearance, Abeta fibrillation, and tangle formation as well as on mitochondrial functions leading to neuronal toxicity and synaptic damage. This review summarizes the pathological cross talk between ApoE and Abeta peptide in Alzheimer's disease. Lastly, we examine emerging gene-based therapeutic approaches encompassing the use of ApoE and their potential opportunities to preventing or treating Alzheimer's disease.

ApoE gene delivery inhibits severe hypercholesterolemia in newborn ApoE-KO mice.

Apolipoprotein E, a key regulator in cholesterol-rich lipoprotein metabolism, is considered a strong candidate for treating hypercholesterolemia and cardiovascular disease. Inherited deficiency of this protein results in type III hyperlipoproteinemia in humans. ApoE-knockout mice, which develop spontaneous hypercholesterolemia, are an excellent model of human atherosclerosis. Here we investigated the therapeutic effects of a plasmid vector encoding human APOE3 sequence intramuscularly injected in hypercholesterolemic newborn mice at the ages of 5 and 14 days. We further explored the possibility of inducing tolerance in newborns when injected early. Our data show that direct i.m. naked DNA injection reduces severe hypercholesterolemia in newborn mice. Moreover, when naked DNA is administrated early, no immune response is generated against the human APOE, allowing repeated administrations. Neonatal therapies are important for the treatment of many genetic childhood diseases where early administration is required to prevent developmental damage. We propose the use of direct i.m. naked gene transfer in newborns to prevent long-term damages arising from hypercholesterolemic conditions.