Nucleic acid vaccines present a compelling alternative to conventional vaccines. Unlike traditional vaccines, nucleic acid vaccines enable the delivery of multiple antigens simultaneously, and thus encoding various antigens to stimulate a broader T-cell response in antigen-presenting cells (APCs). This approach not only enhances immune recognition but also reduces the risk of protein or virus-related contaminations during production, thereby minimizing patient complications and side effects (Ho et al., 2021; Kulkarni et al., 2021; Pardi et al., 2020). mRNA vaccines, in particular, demonstrate unique advantages over DNA vaccines, including higher protein expression, reduced toxicity, and streamlined, scalable manufacturing (Pardi et al., 2018; Jahanafrooz et al., 2020; Park et al., 2021; Chaudhary et al., 2021). Although mRNA is inherently unstable, chemical modifications (Kim S. C. et al., 2022) and advanced delivery vehicles such as LNPs help regulated its in vivo half-life and mitigate degradation.

Furthermore, unlike DNA or viral vaccines, mRNA does not integrate into the genome, a key safety feature that significantly lowers the risk of insertional mutagenesis (Campillo-Davo et al., 2018; Mancianti et al., 2021). Additionally, mRNA is a minimal genetic vector, which can help evade anti-vector immunity and therefore, enable repeated administration (McKinlay et al., 2018). These attributes coupled with LNPs’ proven efficiency in cellular internalization and endosomal escape (Hou et al., 2021; Kon et al., 2022; Tenchov et al., 2021) makes mRNA-LNP platforms particularly appealing for gynecologic malignancies such as ovarian and breat cancers.

Tumor-specific immune responses can be stimulated by exploiting surface antigen differences between tumor cells and normal cells. Unlike infectious diseases, cancer vaccines are used therapeutically to present specific antigens to the host cells (Yaddanapudi et al., 2013; Nahas et al., 2018). While traditional protein/peptide antigens for cancer vaccines have not been successful, mRNA vaccines have shown promise in anti-cancer treatments. mRNA vaccines can express tumor-associated antigens in antigen-presenting cells (APCs) during vaccination, therefore activating APCs and triggering innate/adaptive immune responses to inhibit tumor development and progression. As mentioned, Table 1 depicts some of the current clinical trials targeting gynecological malignancies using RNA-LNP therapies. It is worth noting that although these therapy strategies are not limited to the vaccine approach, we can observe that the prevention and immune potentiation through RNA vaccines are among the commonly used and promising methods. Below, we review a few of the preclinical and clinical studies for the most prevalent gynecological cancers using RNA therapies.

In addition to mRNA-based vaccines, LNPs have also been explored for delivering siRNA in several gynecologic cancers. Several lipid nanocarrier systems are undergoing or have completed clinical trials to assess their safety, efficacy, and translation potential. For instance, ALN-VSP02 (Alnylam Pharmaceuticals) formulation combines kinesin spindle protein (KSP) and vascular epithelial growth factor (VEGF)-siRNA encapsulated in LNP system. A phase I trial enrolled 30 patients with advanced solid tumors, administering intravenous doses of ALN-VSP02 in dose-escalation (0.1–1.5 mg/kg). No major safety concerns were observed, and notably, one patient with endometrial cancer and hepatic metastasis achieved a complete response after 50 doses of ALN-VSP02 (Tabernero et al., 2013a). A dose of 1.25 mg/kg every 2 weeks was recommended for subsequent Phase II evaluations [NCT00882180 (Cervantes et al., 2011; Tabernero et al., 2013b)].

Atu027 (Silence Therapeutics) is yet another siRNA-based LNP formulation designed to knockdown protein kinase N3 (PKN3). In a Phase Ib/IIa (AtuPLEX) involving patients with advanced solid tumors (Strumberg et al., 2012), Atu027 was administered as a single agent and in combination with other anticancer therapies. It was proven to be well-tolerated up to 0.180 mg/kg, with dose-dependent increases in siRNA antisense strand concentrations in plasma. Dose escalation is ongoing for this study.

DCR-MYC or DCR-M1711 (Dicerna Pharmaceuticals), is a Dicer substrate siRNA (DsiRNA) encapsulated in EnCore^™ LNP to downregulate c-Myc across multiple solid tumors. Phase Ib/II trial demonstrated a favorable safety profile at various doses, with some showing promising tumor shrinkage after one cycle of 0.1 mg/kg dose (Tolcher et al., 2015). Despite encouraging initial results, the trial was terminated due to dose-limiting toxicities and sponsor decisions (Harrington et al., 2021).

EPHARNA, also referred to as EphA2-siRNA-DOPC (NCI) formulated by encapsulation of EphA2-siRNA within dioleoylphosphatidylcholine (DOPC) liposoms to reduce EphA2 expression in tumors such as breast and ovarian cancers. When tested in mice, single-dose EPHARNA administration showed minimal toxicity or any pathologic or dose-related microscopic findings in acute or recovery phases. Similarly, minimal infiltration of mononuclear cells was found in major organs when tested in Rhesus macaques. This formulation entered a phase I trial to be tested intravenously twice weekly in recruited patients with advanced metastatic solid cancers [NCT01591356, (Landen et al., 2005)].

MicroRNAs (miRNAs) are key regulators of pathophysiological processes in gynecologic malignancies, acting as either oncogenes (oncomiRs) or tumor suppressors. LNPs, in turn, can effectively deliver miRNA therapeutics – alone or in combination – to treat solid tumors, including TNBC. For instance, Hayward et al. developed LNPs with hyaluronic acid (HA)-modified LNPs to deliver tumor suppressor MicroRNA125a-5p in HER2 positive metastatic breast cancer (Hayward et al., 2016). These HA-LNPs could cause the knockdown of the HER2 proto-oncogene in patient-derived metastatic breast cancer cells (21MT-1), thereby impairing essential signaling pathways involving phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) and inhibiting tumor growth.

Similarly, Dinami et al. signified that LNPs incorporating miR-182-3p could reduce telomeric repeat-binding factor 2 (TRF2) expression in patient-derived TNBC xenograft models, significantly impairing tumor progression (Dinami et al., 2023). Additionally, this LNP formulation could cross the blood-brain barrier to address metastatic brain lesions. In another study, Nevskaya and colleagues demonstrated that delivering a combination of three miRNAs; miR-195-5p, miR-520a, and miR-630, using LNPs, could result in downregulation of SOX2, MYC, TERT, and FZD9 genes in human breast cells and inhibit stemness, thus reducing lung metastasis in vivo C57BL/6 mouse model (Nevskaya et al., 2024). Given this, the development of nanotechnology, especially LNPs, that deliver miRNA therapeutics have shown growing promise of miRNA-LNP technology for future clinical applications in gynecologic oncology.

There is broad ongoing research to identify the potential of CRISPR-Cas9 technology in gynecological cancers, thereby laying grounds for its further clinical application. For instance, Tang et al. used phenylboronic acid (PBA)-derived LNPs to achieve cell-selective genome editing in sialic acid (SA)-overexpressing cervical cancer cells (Tang et al., 2019). Delivery of p53 mRNA and CRISPR components successfully knocked out the HPV18E6 gene, significantly inhibiting tumor growth in vitro and in vivo.

Antibody-conjugated LNPs provide an additional route to targeted genome editing using CRISPR/Cas approach. Rosenblum et al. functionalized CRISPR LNPs with an epidermal growth factor receptor (EGFR) antibody, enabling selective uptake into disseminated ovarian tumors. This strategy yielded ∼80% gene editing and extended survival by 80% in an ovarian cancer mouse (OV8-bearing) model (Rosenblum et al., 2020). Zhang et al. advanced the platform by fabricating a multiplexed dendrimer LNP (siFAK + CRISPR PD-L1-LNPs) co-delivering focal adhesion kinase (FAK) siRNA, Cas9 mRNA, and sgRNA to human ovarian cancer cells (IGROV1) (Zhang et al., 2022). In an ovarian cancer xenograft model (ID8-Luc), PD-L1 knockdown and FAK inhibition together significantly reduced metastasis. This may highlight the potential synergy between CRISPR-based gene editing and RNA interference in gynecologic oncology.

Human papillomavirus (HPV) is a non-enveloped DNA virus with over 200 identified serotypes, about 40 of which can infect the anogenital and mucosal lining through sexual contact. The Centers for Disease Control and Prevention (CDC) reports that more than 42 million Americans are infected with high-risk pathogenic subtypes of HPV, with an annual detection of 13 million new infections (About Human Papillomavirus, 2025). Fifteen high-risk HPV subtypes have been identified to cause cervical intraepithelial neoplasia and squamous cell carcinomas (Quinlan, 2021; Balasubramaniam et al., 2019). These high-risk subtypes contribute to various cancers, including cervical, oropharyngeal, anal, penile, vaginal, and vulvar, the majority of these through the action of the E6 and E7 oncoproteins, which alter host cellular functions leading to the development of cancerous cells (Balasubramaniam et al., 2019; Longworth and Laimins, 2004). For instance, cervical cancer, which affects nearly 500,000 women annually and leads to 300,000 deaths worldwide (Cohen et al., 2019), is predominantly caused by HPV and can be prevented through routine screening, enabling healthcare providers to identify and remove pre-cancerous lesions. Additionally, most vaginal and vulvar cancers are caused by HPV. Furthermore, over 90% of anal cancers are HPV-induced and more common in women than men (Cohen et al., 2019).

Extensive research has shown that HPV-positive tumors exhibit high levels of lymphocyte infiltration. This, in turn, provides a strong rationale for immunotherapy to modulate HPV-related carcinogenesis. Various strategies have been developed to activate the adaptive immune system against HPV-infected tumors. These strategies include immune checkpoint blockade, viral antigen recognition through therapeutic vaccines, adaptive cell therapies, and their combinations with radiotherapy or chemotherapeutics. Unfortunately, there are currently no specific therapies available for HPV-induced cancers, such as cervical cancers. Nevertheless, nanoparticle formulations such as gold, polymeric, or lipid nanoparticles have been investigated to eliminate HPV infection or provide curative measures for these cancers (Medina-Alarcon et al., 2017; Rupar et al., 2019).

Targeting the HPV E6/E7 oncoproteins is a promising therapeutic approach for HPV-related cancers. This includes inducing immune cell populations against E6/E7, silencing oncoprotein expression through RNA interference (RNAi), or utilizing genome editing tools like the CRISPR/Cas system to knock down or knock out the E6 and E7 oncogenes. These gene editing approaches can be combined with LNPs to enhance therapeutic efficiency (Xiong et al., 2021; Aghamiri et al., 2020). Kampel et al. demonstrated that anti-EGFR-targeted LNPs carrying anti-E6/E7 siRNAs led to approximately 50% greater cancer regression than the untargeted siRNA-LNPs, thus providing a promising pathway for cancer treatment through oncoprotein elimination (Kampel et al., 2021). Moreover, E6/E7 oncoprotein-encoded mRNA LNPs showed enhanced antitumor efficacy when co-administered with the stimulator of interferon genes (STING) adjuvant systemically (Tse et al., 2021). Additionally, mRNA-LNPs have been developed to deliver mRNA encoding HPV antigens to cervical cancer cells (Sahin et al., 2020; Kranz et al., 2016; Kreiter et al., 2008). This approach induces significant proliferation of cytotoxic lymphocytes and stimulates immune responses against HPV-positive tumors. Also, Da Silva et al. demonstrated that a single low-dose immunization with any of the three developed LNP-based herpes simplex virus type 1 glycoprotein D (gDE7) mRNA vaccines (self-amplifying mRNA, unmodified mRNA, and nucleoside-modified non-replicating mRNA vaccines) resulted in the activation of E7-specific CD8⁺ T cells, generated memory T cell responses to prevent tumor relapses, and destroyed cervical tumors (Ramos da Silva et al., 2023a). Furthermore, combining mRNA vaccines encapsulated in lipid nanocarriers with immune checkpoint inhibition shows potential for improving the clinical efficacy in cervical cancer (Grunwitz et al., 2019).

HIV affects women worldwide, with approximately 53% of the 39.0 million people living with HIV in 2022 being women over 15 years of age (Grunwitz et al., 2019). In addition to medical challenges, women face significant psychosocial burdens, particularly related to preventing mother-to-child vertical transmission (Fauk et al., 2022). HIV is a retrovirus that enters the host by targeting the cluster of differentiation 4 (CD4) receptor on immune cells and binds to co-receptors like C-C chemokine receptor type 5 (CCR5) and C-X-C chemokine receptor type 4 (CXCR4). The virus reverse transcribes its RNA, integrates into the host DNA, and consequently produces viral particles infecting new host cells. Dendritic cells infected with the virus can transmit it to T cells, leading to further virus dissemination. Studies suggest that the R5 strain of HIV exhibits preferential amplification and dissemination (Deeks et al., 2015; Morrow et al., 2007). Transmission of HIV can occur through mucosal surfaces such as rectal, genital, and, less commonly, oral. While vaginal mucus provides some protection against viral entry, the thin mucus lining of the rectum allows direct access to leukocytes (Morrow et al., 2007).

Current HIV therapeutics include five classes of antivirals: entry inhibitors, reverse transcription inhibitors (RTIs; including both nucleoside RTIs and non-nucleoside RTIs), integrase/transfer inhibitors (INSTIs), and protease inhibitors. Standard first-line regimens often combine an INSTI with RTIs, sometimes augmented with CYP3A inhibitors for longevity and efficacy (Gunthard et al., 2016; HIV/AIDS. In, 2023). In cases of HIV with multiple drug resistance, two new antibody-based therapeutics, ibalizumab (a CD4 post-attachment inhibitor) and leronlimab (a CCR5 inhibitor), can be considered. These antibodies are administered intravenously every 7–14 days to complement existing antiviral therapies. FDA-approved antiretroviral drugs disrupt virion replication by targeting different stages of the virus life cycle, including viral genome replication, protein cleavage, packaging, and virus release from cells. Although these regimens suppress viral loads below detectable limits, they cannot eliminate integrated viral genomes in immune cells. To address the issue of the integrated viral genomes, LNPs with gene editing capabilities are being developed (Herskovitz et al., 2021). These LNPs, co-encapsulating Cas9 mRNA and TatDE guide RNAs (gRNAs), can cleave the HIV genome within infected cells through tumor necrosis factor alpha (TNF-α) stimulation, effectively preventing viral outgrowth (Herskovitz et al., 2021). In parallel, mRNA-LNPs conjugated to anti-CD4 antibodies are being explored to selectively target HIV-infected CD4⁺ T cells (Tombacz et al., 2021), while others focus on delivering HIV-1 gp160–encoding mRNA or anti-CCR5 siRNA to prevent viral entry (Saunders et al., 2021), (Mu et al., 2022; Traore et al., 2022). For instance, Pardi et al. showed that following 6 weeks of a single immunization with mRNA-LNP encoding Env gp120, rabbits and rhesus macaques produced anti-gp120 IgG titers, which were further boosted by subsequent immunization (Pardi et al., 2019). Beyond preventive strategies, Pardons et al. illustrated the application of LNPs where the reactivation capacity of Tat mRNA-LNP was demonstrated in CD4⁺ T cells from HIV patients. Combined with panobinostat, it also caused potent latency reversal, enabling multi-omic analysis of the HIV-1 reservoir for future applications (Pardons et al., 2023).

In another example, a novel approach of inducing “broadly neutralizing antibodies” (bnAbs) against the conserved sites of the HIV-1 spike created a significant hope for a long-lasting prevention and treatment of this disease, specifically by using germline targeting method (Liu Y. et al., 2020; Xie et al., 2024). In this strategy, naïve B cells are recruited to the germinal center and by a guided maturation and sequential exposures to the B cells to the designed antigens, they will be activated and the related memory B cells will be generated. These memory B cells can induce the required immune response (bnAbs) when the protection is needed against the virus infection. In a clinical trial, the germline targeting method was evaluated as a proof of the concept through inducing VCR01-class of bnAbs for engineered outer domain germline targeting version 8 (eOD-GT8) 60mer [(Cohen et al., 2023), NCT03547245]. Given the ease of design, scale up, and adaptability of the mRNA to variants, mRNA-LNP can also be a promising candidate to induce the immunogens in germline targets. For instance, Xie et al. reported the efficacy of a mRNA-LNP delivering germline-targeting N332-GT5 and two other booster immunogens (B11 and B16) to immunize mice against HIV-1 virus in BG18^gH mouse model. They found that their prime-boost strategy using mRNA-LNPs could derive affinity maturation in the target B cells and elicit precursors to bnAbs against HIV-1 variants. They concluded that this strategy may resolve some of the major HIV-related vaccine developments challenges (Xie et al., 2024).

Though maternal antibodies provide infants with crucial early immune protection, they can also interfere with de novo infant antibody responses. This challenge is especially pertinent for HIV-1 vertical transmission. To address maternal antibodies-mediated suppression of immune response, mRNA-LNPs vaccines are being explored in preclinical models. Willis et al. (Willis et al., 2020) reported a nucleoside-modified mRNA-LNP vaccine that could establish prolonged germinal center reaction and partially overcome maternal antibody neutralization in mice pups to induce de novo immune response against influenza virus. They hypothesized that this prolonged immune response could be due to the prolonged antigen availability by mRNA-LNPs leading to stronger germinal center responses.

Despite efforts in antiviral development, no definitive HIV cure or universal prophylaxis exists. Emerging RNA-LNP platforms, bnAbs, and combination therapies may improve patient outcomes by targeting multiple steps in viral pathogenesis, with particular relevance to maternal and infant health (Landovitz et al., 2023).

Osteoporosis is a common disorder characterized by low bone mass, deterioration of bone microarchitecture, and increased risk of brittle fracture (NIH, 2001). The imbalance between bone formation and resorption is more prevalent in postmenopausal women, leading to heightened susceptibility to fractures. Current anti-osteoporosis treatments primarily include hormonal therapies and bisphosphonates, both of which aim to prevent bone resorption or enhance restoration.

Gene therapy offers a promising route for novel osteoporosis treatments. For instance, studies targeting bone marrow mesenchymal stem cells (MSCs) have demonstrated the potential of manipulating endogenous bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs) to promote osteo-induction (Vhora et al., 2019). Vhora et al. (2019) employed ionizable LNPs bearing bone-homing peptides to deliver plasmid-encoded BMP-9, thereby stimulating osteoblastic lineage cells. In another study, Basha et al. (2022) used LNPs encapsulating siRNA to silence the suppressor gene GNAS, facilitating bone restoration. Also, Xue and colleagues built upon these concepts by developing a series of bisphosphonate (BP) lipid-like materials to identify an BP-LNP formulation 490BP-C14. This formulation improved mRNA expression and localization in the in vivo bone microenvironment when tested in mice (Xue et al., 2022). Intravenous administration of 490BP-C14 resulted in therapeutic bone morphogenetic protein-2 secretion from the bone microenvironment. Such targeted LNP systems could complement current therapies by preventing bone loss and enhancing new bone formation in women diagnosed with osteoporosis.

Non-coding RNAs – such as RNA interference (RNAi), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs) – have also demonstrated therapeutic potential for managing osteoporosis (Ko et al., 2020; Silva et al., 2019). CircRNAs are particularly interesting because of their unique structure, which makes them less prone to degradation than non-circular RNAs (Enuka et al., 2016). They have been shown to serve as biomarkers for the treatment of osteoporosis by promoting osteogenesis (Huang et al., 2019; Zhao et al., 2018; Yu and Liu, 2019; Hansen et al., 2013; Chen et al., 2019; Gu et al., 2017). In this regard, LNPs, as the delivery vehicle, can improve RNA therapy for osteoporosis by enhancing its efficacy by successfully delivering the RNA cargo to the desired cell lineages (Basha et al., 2016; Hallan et al., 2022).

Endometriosis is caused by the ectopic endometrial tissue dispersion and growth outside the endometrium (Kabani et al., 2022). It affects roughly 10% of women of reproductive age, causing pain and infertility with a considerable impact on quality of life (Giudice and Kao, 2004; Culley et al., 2013). The currently available treatments include surgical interventions and medications such as nonsteroidal anti-inflammatory drugs (NSAIDs) and hormonal therapy using progestins (Vercellini et al., 2011; Becker et al., 2017). However, these treatments have several side effects and are not always effective in women who plan to conceive (Vercellini et al., 2011). Therefore, there is a growing need for nonhormonal, safe, and effective therapies that can target endometriosis by inhibiting the development of new lesions (Adams et al., 2017).

Non-coding RNAs (ncRNAs), such as miRNAs and lncRNAs, are among the non-conventional therapeutic tools for gene regulation and potential treatment of endometriosis (Adams et al., 2017; Evans et al., 2016). Many studies have demonstrated that miRNAs significantly regulate gene expression in endometriosis (Panir et al., 2018; Teague et al., 2010). Preclinical studies in murine models have shown that the therapeutic delivery of miRNAs, such as Let-7b and miRNA-142-3p, can effectively suppress endometriotic lesions via downregulation of various genes that have a role in endometriosis, including Kruppel-like factor 9 (KLF9) and vascular endothelial growth factor A (VEGF-A) signaling (Sahin et al., 2018; Ma et al., 2019). Moreover, miRNAs can modulate endometrial inflammation by regulating pro-inflammatory cytokines, such as TNF-α, interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) in patients with endometriosis (Nematian et al., 2018).

LncRNAs likewise influence transcription and translation, with aberrant expression linked to endometriosis onset (Yan et al., 2020). For example, downregulation of the lncRNA H19 reduces endometrial cell dissemination via the H19/let-7/insulin-like growth factor 1 receptor (Igf1r) pathway (Ghazal et al., 2015). Another study demonstrated that a lncRNA (LINC00261) could inhibit the growth and proliferation of endometriotic cells (Sha et al., 2017).

In parallel, nanoparticle-based delivery strategies have shown promise for targeting endometriotic cells. Bedin et al. combined a low-density lipoprotein (LDL)-like nanoemulsion loaded lipid nanoparticles (LDE) with a chemotherapeutic agent to target endometrial cells and prevent significant systemic side effects of chemotherapy (Bedin et al., 2019). A significant uptake of LDE was observed in the endometriotic foci to enhance the consumption of LDL by these endometrial cells. Building on these findings, RNA-LNP systems designed to modulate key regulatory RNAs may offer an alternative therapeutic approach for endometriosis.

Uterine fibroids (leiomyomas) are the most common benign uterine tumors in reproductive-age women, impacting up to 70% over their lifetimes (Baird et al., 2003; Wise and Laughlin-Tommaso, 2016; Al-Hendy et al., 2017; Yang et al., 2022). Although benign, fibroids can cause extensive symptoms, including heavy menstrual bleeding, pelvic discomfort, and infertility. Standard treatments – ranging from surgery and hormonal therapies (e.g., gonadotropin-releasing hormone or GnRH, agonists) to uterine artery embolization – often carry side effects or risks that necessitate the search for safer alternatives (Yang et al., 2022). While published research on RNA-LNPs for fibroid treatment is limited, other nanoparticle approaches have demonstrated feasibility for delivering therapeutic nucleic acids. For instance, a peptide-based nanoparticle targeting αvβ3 integrin transported a suicide gene (HSV-TK) to uterine leiomyoma cells, increasing cytotoxicity with ganciclovir (Egorova et al., 2022). Under a similar strategy, the researchers demonstrated that a peptide-based magnetic nanoparticle formulation could promote the delivery of plasmid DNA to uterine leiomyoma cells and induce cell death after sequential GCV treatments (Shtykalova et al., 2022). Given LNPs’ favorable safety profiles and proven clinical success, their application in uterine fibroid therapy – especially for gene-based or immunotherapeutic approaches – holds potential. Indeed, immunotherapy is an emerging area of investigation, as evidenced by a Phase II trial using inactivated myoma tissue and blood samples for oral vaccination (NCT03550703). By leveraging tumor-specific or personalized antigenic targets, mRNA-LNP vaccines akin to those developed for certain gynecologic cancers may represent a future direction for fibroid management.

Congenital disorders involve structural or functional abnormalities that arise prenatally, often causing significant morbidity and mortality. Existing interventions – such as fetal surgery, in utero gene therapy, and in utero stem cell transplantation (IUSCT) – frequently pose high risks (Swingle et al., 2023a; Palanki et al., 2021). Amongst these, in utero gene therapy offers advantages, including being minimally invasive and achieving sustained phenotype correction for many genetic diseases. Recent advances have explored in utero mRNA-LNP delivery to treat or prevent congenital conditions. Swingle and co-workers developed stable mRNA-LNPs for intra-amniotic delivery to show potent mRNA delivery in vitro and in utero in a murine model (Swingle et al., 2022). They used the orthogonal design of experiments (DOE) to screen a space of 256 possible LNP formulations. They identified that more stable mRNA-LNPs have higher in utero mRNA delivery than unstable LNPs. Therefore, developing this stable ionizable RNA-LNP formulation in amniotic fluid paved the way for screening potential LNPs designed for prenatal delivery to treat congenital diseases in pregnant women. This group also formulated VEGF-A mRNA-LNPs for delivery to the placental cells, including trophoblasts, endothelial cells, and immune cells, to achieve placental vasodilation that would benefit maternal and fetal health (Swingle et al., 2023b).

Another research group also built a library of ionizable LNPs for in utero mRNA delivery to mouse fetuses that resulted in the accumulation with the livers, lungs, and intestines with higher efficiency and safety as opposed to DLin-MC3-DMA and jetPEI^®-based delivery systems (Riley et al., 2021). There have also been attempts to target organs like the heart, kidneys, lungs, and gastrointestinal tract via in utero delivery of LNPs. Gao et al. demonstrated that using mRNA-LNP complexes, 0.64%–12.4% of the cells in the lungs, heart, liver, kidneys, brain, and gastrointestinal tract were transfected with low toxicity. Additionally, LNPs complexed with Cas9 mRNA and sgRNA could edit 0.2%–0.8% of cells in the liver, heart, gastrointestinal tract, and kidneys of Ai9 mice following in utero delivery (Gao et al., 2023). This extrahepatic targeting could be leveraged to address various congenital diseases in pregnant women.

Furthermore, hereditary angioedema (HAE), a rare genetic disorder with a strong female predominance, exemplifies how RNA-LNP delivery might also combat rare pregnancy-aggravated conditions (Caballero et al., 2014). For example, NTLA-2002, a CRISPR/Cas9 LNP platform, was developed by Intellia Therapeutics^® to target the kallikrein B1 (KLKB1) gene. Its single administration resulted in ∼70% KLKB1 gene editing and >90% reduction of kallikrein protein when tested in mice and monkeys. NTLA-2002 is undergoing a Phase II clinical trial in recruited HAE patients (NCT05120830).

The diverse applications of RNA-LNP therapeutics in women’s health, from gynecologic cancers and viral infections to osteoporosis and congenital disorders, underscore the immense potential of this platform. Nevertheless, the successful translation of RNA-LNPs into clinical practice hinges on overcoming multiple challenges – particularly those related to targeted delivery, safety, and long-term efficacy. The following section explores these obstacles and discusses emerging strategies to optimize RNA-LNPs for women’s health applications.

A straightforward way to bypass hepatic accumulation of LNPs is to change the administration route from intravenous delivery to other administration routes, which could shorten the delivery path and enhance accessibility to the target cells (Figure 3) (Chenthamara et al., 2019; Kim J. et al., 2022; Zimmermann et al., 2022; Leong and Ge, 2022; Patel et al., 2019; Kumbhar et al., 2022; Bardoliwala et al., 2021). For example, intra-tumoral injection has been utilized to deliver LNPs and could slow down tumor progression or even eliminate established tumors (Liu et al., 2022; Li et al., 2020; Hsu et al., 2013). In one study on a TNBC model, a tumor acidity-responsive nanoparticle released chemokine C-C motif ligand 25 (CCL25) protein and cluster of differentiation 47 (CD47) siRNA sequentially after intra-tumoral administration, and therefore enhanced immunotherapy against the cancer cells by promoting CCR9+ CD8⁺ T cell tumor infiltration (Chen et al., 2020).

Additionally, intramuscular (IM) administration has been chosen for mRNA vaccine development. This approach can shorten the route for LNPs to lymph node clusters and enhance immune responses (Miao et al., 2021; Hassett et al., 2019). Zika virus is of particular concern to women’s health because it affects them at nearly twice the rate of men and can be transmitted from a pregnant woman to her fetus. Richner et al. developed the modified mRNA vaccines with LNP, successfully preventing Zika virus disease through IM delivery. One concern for Zika virus mRNA therapy is that the produced antibody against Zika may cross-react with the Dengue virus and enhance its infection in cells. However, Richner et al. have shown that the mRNA-LNP approach reduced cross-reaction risk and sensitized individuals to subsequent exposure to Dengue virus in mice (Richner et al., 2017). Furthermore, clinical trials for mRNA-1893 and mRNA-1325 from Moderna are underway to combat the Zika virus (Essink et al., 2023).

The intraperitoneal (IP) route of administration has also been explored for targeted gene editing using LNPs as delivery systems in gynecological cancers such as ovarian cancer. For instance, Rosenblum and Gutkin et al. developed EGFR-targeted CRISPR-LNPs against PLK1 (sgPLK1-cLNPs) that led to selective uptake into disseminated ovarian cancer cells and facilitated up to ∼80% gene editing in vivo, significantly inhibiting tumor growth and improving survival by 80% in a mouse model (Rosenblum et al., 2020).

Another method of the local delivery in female disorders is the intravaginal administration. Using vaginal delivery, the cargos avoid liver first-pass clearance and systemic dilution effects and can be used for uterovaginal viral infections and gynecologic cancers. Upon vaginal administration, the first uterine pass effect (Campana-Seoane et al., 2019; De Ziegler et al., 1997) allows preferential drug accumulation in the uterus and vagina through counter-current drug exchange between the blood in the vaginal veins and the uterine artery. Vagina tissues have a high surface area with high blood flow in the muscular layers, which helps improved drug absorbance at this route. Also, it has been reported that adding polyethylene glycol to the surface of liposomes can enhance tissue penetration of intravaginal delivery against HPV infection in the ex vivo sheep vaginal epithelium model (Joraholmen et al., 2017). However, unlike the successful application of intravaginal delivery of small molecules, for macromolecules such as RNAs, the thick mucosal layer of vagina and fast clearance of the mucous layer limit the delivery. Additionally, the delivery of RNA molecules through this route faces another challenge due to the low pH (3.5–4.5) of the vagina’s surface (Kim et al., 2018). Since RNA-LNPs are a self-assembled and pH-dependent structure, the low pH of the vaginal environment may significantly affect the stability of the particles and their efficacy.

In fetal congenital disorders, in utero delivery can be a valuable option for gene delivery (Swingle et al., 2022; Riley et al., 2021; Ullrich et al., 2021). It can be accomplished either by injection in the vitelline vein (which provides direct access to the fetal liver) or through intra-amniotic fluid (Palanki et al., 2021; Swingle et al., 2022; Ullrich et al., 2021). For example, in a study by Riley et al., different LNPs were screened for luciferase mRNA delivery via mouse vitelline vein injection (Riley et al., 2021). They identified formulations that efficiently could accumulate within fetal livers, lungs, and intestines compared to DLin-MC3-DMA and jetPEI. They observed that the selected LNPs could deliver mRNAs and induced significant expression of the target protein in the liver. Also, from the same scientific group, Swingle et al. engineered a library of LNPs using orthogonal design of experiments to evaluate the effect of LNP structure on their stability in amniotic fluid and to enable intra-amniotic mRNA delivery in utero (Swingle et al., 2022). Their study found multiple lead LNPs that were stable in ex utero amniotic fluids of different animal species and in humans. Also, they showed that these ex-vivo screened LNPs enabled effective mRNA delivery in primary fetal lung fibroblast cell culture and in utero by intra-amniotic injection in a murine model.

Table 2 depicts selected studies on actively targeted RNA-LNPs for women’s health. Although local delivery bypasses many systemic barriers, IV administration remains vital for widespread treatment of women’s diseases. Under these conditions, targeted LNPs must traverse the bloodstream and overcome organ-specific barriers en route to non-liver tissues (Figure 3, and (Mitchell et al., 2021; Whitehead et al., 2009; Yin et al., 2014). There are some challenges and opportunities that targeted delivery in female patients need to be considered when applying the targeted RNA-LNP therapy. Below, we first discuss targeted RNA-LNP delivery as a general term. Then, we give a few brief examples of delivery for women’s diseases by RNA-LNPs.

As a general term, the targeted LNP delivery system must: 1) prevent glomerular filtration, phagocytosis, and degradation of nucleic acid therapeutics in the kidney, liver, and bloodstream; 2) cross the vascular endothelial barrier and extracellular matrix; and 3) enter targeted cells, escape endosomes, and release loaded nucleic acids. Successful retargeting of LNPs to non-liver sites first requires de-targeting from the liver, and thus extending the blood circulation time. Immune-invisible coatings like PEGylation (Suk et al., 2016), inert polymeric coatings (Debayle et al., 2019; Chen et al., 2021), cell membrane coating (Fang et al., 2018; Luk and Zhang, 2015), and “self” peptides (Khan et al., 2022; Oltra et al., 2014) resist serum protein binding, reducing clearance by the reticuloendothelial system (RES). Elongated circulation time increases the likelihood of LNPs encountering targeted cells. Saunders et al. (Saunders et al., 2020) developed an effective method to minimize LNP capture by liver cells. By pretreating with a “nanoprimer” before LNPs loaded with RNA therapeutics, the bioavailability of RNA-loaded LNPs significantly improved (Saunders et al., 2020). Ouyang et al. (Ouyang et al., 2020) discovered a threshold dose that overwhelms Kupffer cell uptake rates. Above this threshold, liver clearance of nanoparticles nonlinearly decreases, extending circulation time (Ouyang et al., 2020).

Researchers have made significant advancements in re-targeting strategies by exploring the potential of passive and active targeting techniques/engineering (Figure 3) for redirecting LNPs toward desired targets (Dilliard and Siegwart, 2023).

Although mRNA vaccines can generate rapid protein expression, their short half-life may limit applications requiring longer-term protein production. Self-amplifying RNA (saRNA) was developed to address this challenge (Figure 2). By incorporating a viral replicon encoding nonstructural genes, saRNA constructs can amplify themselves intracellularly, prolonging protein expression and enhancing immunogenicity compared to conventional mRNA (Blakney et al., 2021; Bloom et al., 2021). Lower doses of saRNA-LNPs can thus achieve higher immune responses, offering potential in both prophylactic vaccines and therapeutic applications demanding substantial protein expression.

Nevertheless, presence of a viral replicon in the saRNA structure significantly increases its size by thousands of bases to pose stability issues. Also, these nucleotides that are incorporated into the saRNA structure must be compatible with the T7 RNA polymerase and cellular translation machinery, as well as the viral nonstructural genes that are necessary for RNA amplification and subsequent protein expression (Maruggi et al., 2019). To resolve this issue, Aziz et al. utilized noncanonical nucleotides, such as 5-methylcytidine (m5C) and 5-methyluridine (m5U), that were found to be compatible with standard mRNA translation and reduced the immune stimulation and recognition of RNA molecules by Toll-like receptors (TLRs) (Azizi et al., 2024). Hence, the use of such modified nucleotides with saRNA-based LNPs displayed great potential for a variety of therapeutic applications. Similarly, another group of researchers developed an saRNA-LNP vaccine to demonstrate substantially increased immunogenicity in comparison to the unformulated RNA (Geall et al., 2012). Ramos da Silva et al. explored the potential of saRNA-LNP as a cancer vaccine for human papillomavirus-associated cancers. It was shown that a single dose of an saRNA-LNP vaccine encoding a fusion protein of the type 1 herpes simplex virus (HSV-1) glycoprotein D (gD) and the HPV-16 E7 oncoprotein (E7) could induce robust immune response to control advanced tumor progression in preclinical settings (Ramos da Silva et al., 2023b).

Despite these advances, concerns remain regarding the replicative nature of such vaccines, particularly in pregnant women where viral replicons could theoretically affect fetal development (Comes et al., 2023). However, studies on attenuated YFV17D vector vaccines suggest that, while replicons can cross the placenta, they may not necessarily harm embryonic growth (da Silva et al., 2020). Additional preclinical and clinical studies are still required to implement saRNA-LNP based vaccines in vulnerable individuals.

Circular RNAs (circRNAs) are a novel vaccine platform in which the RNA is covalently closed without free ends, increasing resistance to enzymatic degradation (Figure 2). Translation of circRNAs relies on internal ribosome entry sites (IRES) or m⁶A modifications, leading to higher protein expression and a longer half-life than linear mRNA counterparts (Bai et al., 2022; Wesselhoeft et al., 2018). Even low doses of circRNA formulations can generate robust immune responses, making them attractive candidates for next-generation vaccine development. For instance, CircRNA^RBD and VFLIP-X have shown potent immunogenicity against SARS-CoV-2 in preclinical models (Qu et al., 2022; Seephetdee et al., 2022). Moreover, a circRNA^OVA-luc-LNP (OVA[257-264]-luciferase-coding circRNA) vaccine was also constructed by a group of researchers to show anti-tumor immune response in a wide range of difficult-to-treat malignancies (Li et al., 2022).

Despite these promising results, the clinical translation of circRNA-LNP formulations is yet limited by various challenges encountered in their development, manufacturing, quality control, and safety. Designing circRNA-LNP vaccines to optimize self-adjuvant effects, improve circularization efficiency, and refine fragment length remains a key step for maximum efficacy (Chen et al., 2023). Additional safety concerns center on that fact that exogenous circRNAs may disrupt the biological functions of naturally present circRNAs in the body following administration. Noticeably, it has been shown that circRNAs are present in granulosa cells in ovarian follicles, placenta, several different fetal tissues, and maternal blood in pregnant women (Arthurs et al., 2022). It has also been proven that circRNAs are involved in a variety of biological processes that are related to the pathogenesis of pre-eclampsia during pregnancy (Liao et al., 2024). Given this, administration of circRNA-LNP formulations in pregnant women can also result in unnecessary complications. Also, circRNA-LNP vaccines contain residual dsRNA content, which could result in unwanted immunogenicity or myocarditis after vaccination (Han et al., 2021; Singh et al., 2021).

Although little research exists specifically for circRNA-LNPs in gynecologic cancers, expanding these studies could illuminate their therapeutic potential while addressing the unique safety requirements of pregnant women. Technological advancements that address current delivery and formulation challenges will be necessary to fully exploit circRNA vaccines and therapeutics against infections and malignancies.

Preclinical models are indispensable for understanding the therapeutic mechanisms, safety, and efficacy of RNA-LNP interventions in women’s diseases. Larger species such as bovine, porcine, and ovine models can mimic human physiology in specific contexts, but mice remain the most common due to their availability, cost-effectiveness, and well-characterized genetics. For the purposes and scope of this review, we are focusing on the latter category. Table 3 briefly overviews some mouse models used to study various women’s diseases. Murine models fall into two main categories: (i) genetically modified mice that replicate molecular pathways relevant to human disease, and (ii) chemically or surgically induced models. While these systems afford invaluable mechanistic insights, they do not always recapitulate the complexity of human pathology. For instance, the keratinized mouse vaginal epithelium – with thickness fluctuating across estrous phases – limits translational relevance for human vaginal drug delivery (McCracken et al., 2021). Some gynecological conditions are challenging to mimic in mice models mainly because of their short duration and lack of spontaneous incidence in mice. For instance, endometriosis at the late stages requires that mice be evaluated for months to a year, which makes it very hard to keep the animal alive for such a duration. Additionally, only non-human primate species have spontaneous endometriosis, and other animals, including mice, lack this capability (Lagana et al., 2018). Similarly, most murine models of ovarian and cervical cancers cannot accurately capture recurrent malignancies or drug resistance. Moreover, placental differences (haemotrichorial in mice vs. haemomonochorial in humans) complicate direct extrapolation to pregnancy-related conditions (Furukawa et al., 2014).

Despite these constraints, innovations in genetic engineering and immunocompromised “humanized” mice can bring preclinical studies closer to clinical reality. Nevertheless, investigators must remain cautious when inferring therapeutic outcomes for women’s health. Mouse data, while foundational, should be integrated with complementary in vitro and in silico studies, as well as larger, more physiologically analogous animal models, to robustly predict clinical responses.

Recent research has explored the possible benefits of using nanoparticles (including LNPs) in gynecological and pregnancy-related disorders (Bedin et al., 2019; Riley et al., 2021; Michy et al., 2019; Liu et al., 2017; Zhai et al., 2018). Such systems could be used for targeted drug delivery to the female reproductive system in both pregnant and non-pregnant women, improving treatment outcomes (Riley et al., 2021; Sharma et al., 2021; Young et al., 2022) and even serve as a theranostic platform in gynecological disorders (Moses et al., 2020). Despite these benefits, detailed knowledge of nanoparticle risk/benefit profiles in gynecology, pregnancy, and lactation remains limited, largely due to the complexity of female reproductive physiology and ethical restrictions on clinical testing (Sharma et al., 2021).

The female reproductive system is dynamic and undergoes significant changes during pregnancy and lactation, making it essential to understand the interactions between nanoparticles and the maternal-fetal interface, as well as their potential impact on fetal and neonatal health (Sharma et al., 2021; de Araujo et al., 2020; Grafmuller et al., 2013). Ethical considerations further restrict data availability, particularly regarding potential risks to pregnant women and their fetuses. Current knowledge gaps include whether nanoparticle exposure affects ovulation, implantation, embryo development, and hormonal balance (Ajdary et al., 2021; Ahmad, 2022), and how placental accumulation might impair fetal growth or health (Ahmad, 2022). Having said that, this direct impact of NPs is not always the only issue in pregnancy applications. The other deriving fetal toxicity may come through the maternal toxic response to the administered dose that can be vertically transmitted through the placenta and impact the fetus with toxicity and abnormal growth or teratogenicity (Dugershaw et al., 2020). These concerns largely explain why pregnant women are typically excluded from early drug discovery trials (Mazer-Amirshahi et al., 2014), as witnessed during initial COVID-19 vaccine testing (Prahl et al., 2022; Dangel et al., 2022; Couzin-Frankel, 2022). Recent studies eventually shed some light on the safety of mRNA-LNP vaccines in pregnancy. The available data show that LNP products can be detected in serum for a month after the first dose of the COVID vaccine and a week after the second dose administration. Prahl et al. found that LNPs cannot pass the placenta and were not detected in the fetus, though IgGs against SARS-CoV-2 antigens were detectable in the fetal blood, indicating passive immunization of the fetus (Prahl et al., 2022). While small molecules and certain antibodies (such as IgG but not IgM) generally traverse the placental barrier and reach the neonatal organs, nanoparticles, such as conventional LNP composition (used in mRNA-LNP COVID vaccines), do not unless specially engineered to passively or actively target in utero delivery (Figueroa-Espada et al., 2020).

Another challenge of LNPs is the unclear acute and chronic effects of LNPs and RNA molecules on women’s health. As the treatment with gene therapy modalities, such as mRNA in enzyme replacement therapy, is chronic, accumulating LNPs in tissues over repeated administration may result in adverse health effects. Nevertheless, this may be less crucial for the limited-dosing frequency of gene correction methods, such as CRISPR/Cas, etc. Our knowledge of chronic immune system interaction with LNPs or in vitro-transcribed RNA molecules has yet to mature fully. This potential chronic immunogenicity could be a concern for vulnerable populations such as immune-compromised elderly women or pregnant women. Our understanding of COVID-19 vaccines and RNA delivery by lipids may help to elucidate some of the other individual and sex-related influences and allergic reactions to the LNP components, especially for the maternal and perinatal situation where the mother’s physiology is already at a relatively immune-compromised state (Sharma et al., 2021).

It has been shown that some of the administered lipids in RNA-LNP products can enhance the adjuvant effects of the carrying components and induction of efficient immune response in a useful way for vaccine purposes (Luan et al., 2022). Nonetheless, there is a possibility that an uncontrolled immune response may also lead to unintended consequences, such as inflammation due to reactive oxygen species production (ROS) in the placenta of pregnant women or tissue/organ damage. Furthermore, the potential impact of LNPs and RNA molecules on the reproductive system, particularly post-partum and lactation, requires careful scrutiny. In a survey of lactating mothers who received the COVID vaccine, all participants showed detectable levels of neutralizing antibodies in breastmilk after the second dose of vaccine, and only 2% of the mothers had detectable mRNA-vaccine particles or components in their breastmilk over 1 week post-vaccination (Yeo et al., 2021). This study did not find any sensitivity in newborns after maternal vaccination, and the authors concluded that nursing post-vaccination confers temporary passive immunity (Yeo et al., 2021). Further studies are needed to fully uncover the safety of these therapeutics for both the mother and the developing fetus or breastmilk-fed infant to avoid any potential harm.