Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques.

The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.

Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

SARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody Generation.

The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.

The Transcription Factor T-bet Resolves Memory B Cell Subsets with Distinct Tissue Distributions and Antibody Specificities in Mice and Humans.

B cell subsets expressing the transcription factor T-bet are associated with humoral immune responses and autoimmunity. Here, we examined the anatomic distribution, clonal relationships, and functional properties of T-bet+ and T-bet- memory B cells (MBCs) in the context of the influenza-specific immune response. In mice, both T-bet- and T-bet+ hemagglutinin (HA)-specific B cells arose in germinal centers, acquired memory B cell markers, and persisted indefinitely. Lineage tracing and IgH repertoire analyses revealed minimal interconversion between T-bet- and T-bet+ MBCs, and parabionts showed differential tissue residency and recirculation properties. T-bet+ MBCs could be subdivided into recirculating T-betlo MBCs and spleen-resident T-bethi MBCs. Human MBCs displayed similar features. Conditional gene deletion studies revealed that T-bet expression in B cells was required for nearly all HA stalk-specific IgG2c antibodies and for durable neutralizing titers to influenza. Thus, T-bet expression distinguishes MBC subsets that have profoundly different homing, residency, and functional properties, and mediate distinct aspects of humoral immune memory.

A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice.

SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.

Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.

IL7 increases targeted lipid nanoparticle-mediated mRNA expression in T cells in vitro and in vivo by enhancing T cell protein translation.

The use of lipid nanoparticles (LNP) to encapsulate and deliver mRNA has become an important therapeutic advance. In addition to vaccines, LNP-mRNA can be used in many other applications. For example, targeting the LNP with anti-CD5 antibodies (CD5/tLNP) can allow for efficient delivery of mRNA payloads to T cells to express protein. As the percentage of protein expressing T cells induced by an intravenous injection of CD5/tLNP is relatively low (4-20%), our goal was to find ways to increase mRNA-induced translation efficiency. We showed that T cell activation using an anti-CD3 antibody improved protein expression after CD5/tLNP transfection in vitro but not in vivo. T cell health and activation can be increased with cytokines, therefore, using mCherry mRNA as a reporter, we found that culturing either mouse or human T cells with the cytokine IL7 significantly improved protein expression of delivered mRNA in both CD4+ and CD8+ T cells in vitro. By pre-treating mice with systemic IL7 followed by tLNP administration, we observed significantly increased mCherry protein expression by T cells in vivo. Transcriptomic analysis of mouse T cells treated with IL7 in vitro revealed enhanced genomic pathways associated with protein translation. Improved translational ability was demonstrated by showing increased levels of protein expression after electroporation with mCherry mRNA in T cells cultured in the presence of IL7, but not with IL2 or IL15. These data show that IL7 selectively increases protein translation in T cells, and this property can be used to improve expression of tLNP-delivered mRNA in vivo.

Use of HSC Targeted LNP to Generate a Mouse Model of Lethal α-Thalassemia and Treatment via Lentiviral Gene Therapy.

-Thalassemia (AT) is one of the most commonly occurring inherited hematological diseases. However, few treatments are available, and allogeneic bone marrow transplantation (BMT) is the only available therapeutic option for patients with severe AT. Research into AT has remained limited due to a lack of adult mouse models, with severe AT typically resulting in in utero lethality. By using a lipid nanoparticle (LNP) targeting the receptor CD117 and delivering a Cre mRNA (mRNACreLNPCD117), we were able to delete floxed -globin genes at high efficiency in hematopoietic stem cells (HSC) ex vivo. These cells were then engrafted in the absence or presence of a novel α-globin expressing lentiviral vector (ALS20I). Myeloablated mice transplanted with mRNACreLNPCD117-treated HSC showed a complete knockout of -globin genes. They demonstrated a phenotype characterized by the synthesis of hemoglobin H (-tetramers,  or HbH), aberrant erythropoiesis, and abnormal organ morphology, culminating in lethality approximately eight weeks following engraftment. Mice receiving mRNACreLNPCD117-treated HSC with at least one copy of ALS20I survived long-term with normalization of erythropoiesis, decreased the production of HbH, and ameliorated the abnormal organ morphology. Furthermore, we tested ALS20I in erythroid progenitors derived from -globin-KO CD34+ and cells isolated from patients with both deletional and non-deletional HbH disease, demonstrating improvement in -globin/-globin mRNA ratio and reduction in the formation of HbH by HPLC. Our results demonstrate the broad applicability of LNP for disease modeling, characterization of a novel severe mouse model of AT, and the efficacy of ALS20I for treating AT.

Anti-PfGARP activates programmed cell death of parasites and reduces severe malaria.

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.

Throughput-scalable manufacturing of SARS-CoV-2 mRNA lipid nanoparticle vaccines.

Lipid nanoparticles (LNPs) are a potent delivery technology that have made it possible for the recent clinical breakthroughs in mRNA therapeutics and vaccines. A key challenge to the broader implementation of mRNA therapeutics and vaccines is the development of technology to produce precisely defined LNP formulations, with throughput that can scale from discovery to commercial manufacturing and meet the stringent manufacturing standards of the pharmaceutical industry. To address these challenges, we have developed a microfluidic chip that incorporates 1×, 10×, or 256× LNP-generating units that achieve scalable production rates of up to 17 L/h of precisely defined LNPs. Using these chips, we demonstrate that LNP physical properties and potency in vivo are unchanged as throughput is scaled. Our chips are fabricated out of silicon and glass substrates, which have excellent solvent compatibility, compatibility with pharmaceutical manufacturing, and can be fully reset and reused. SARS-CoV-2 mRNA-LNP vaccines formulated by our chips triggered potent antibody responses in a preclinical study. These results demonstrate the feasibility of directly translating microfluidic-generated LNPs to the scale necessary for commercial production.

mRNA-based therapeutics: looking beyond COVID-19 vaccines.

Recent advances in mRNA technology and its delivery have enabled mRNA-based therapeutics to enter a new era in medicine. The rapid, potent, and transient nature of mRNA-encoded proteins, without the need to enter the nucleus or the risk of genomic integration, makes them desirable tools for treatment of a range of diseases, from infectious diseases to cancer and monogenic disorders. The rapid pace and ease of mass-scale manufacturability of mRNA-based therapeutics supported the global response to the COVID-19 pandemic. Nonetheless, challenges remain with regards to mRNA stability, duration of expression, delivery efficiency, and targetability, to broaden the applicability of mRNA therapeutics beyond COVID-19 vaccines. By learning from the rapidly expanding preclinical and clinical studies, we can optimise the mRNA platform to meet the clinical needs of each disease. Here, we will summarise the recent advances in mRNA technology; its use in vaccines, immunotherapeutics, protein replacement therapy, and genomic editing; and its delivery to desired specific cell types and organs for development of a new generation of targeted mRNA-based therapeutics.

Hepatitis C virus modified sE2F442NYT as an antigen in candidate vaccine facilitates human immune cell activation.

The rational selection of hepatitis C virus (HCV) vaccine antigen will aid in the prevention of future chronic liver disease burden and associated healthcare costs. We have previously shown that HCV E2 glycoprotein is not highly immunogenic, and the modification of E2 reduced CD81 binding and displayed altered cytokine and protective immune responses in vitro and in a surrogate mouse model. Here, we compared the influence of a parental and a modified sE2F442NYT glycoprotein region from HCV genotype 1a for the activation of peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), CD4+T cells, and B cells. Modified sE2F442NYT, when incubated with DCs, induced a higher number of CD86-positive cells. The sE2F442NYT or parental sE2 encapsulated as mRNA-lipid nanoparticle (sE2F442NYT mRNA-LNP) primed DCs co-cultured with autologous CD4+T cells did not induce CD25 or forkhead box P3 expression. PBMC-derived CD4+T cells treated with sE2F442NYT exhibited enhanced signal transducer and activator of transcription (Stat)1/Stat4 phosphorylation in response to anti-CD3/CD28 stimulation in comparison to parental sE2 treatment and facilitated isotype switching in B cells, leading to the generation of a broader subclass of antibodies. Cells treated with modified sE2F442NYT displayed an increase in activated Stat3 and extracellular signal-regulated kinase (ERK). Likewise, PBMC-derived naïve B cells upon in vitro stimulation with sE2F442NYT induced an increased proliferation, Stat3 and ERK activation, and protein kinase B (Akt) suppression. Thus, the modified sE2F442NYT antigen from HCV facilitates improved DC, CD4+T, and B cell activation compared to parental sE2 to better induce a robust protective immune response, supporting its selection as an HCV candidate vaccine antigen for preclinical and clinical HCV vaccine trials.IMPORTANCEThe nature of an enhanced immune response induced by sE2F442NYT will help in the selection of a broad cross-protective antigen from hepatitis C virus genotypes, and the inclusion of relatively conserved sE1 with sE2F442NYT may further strengthen the efficacy of the candidate vaccine in evaluating it for human use.

The Regulation of Nucleic Acid Vaccine Responses by the Microbiome.

Nucleic acid vaccines, including both RNA and DNA platforms, are key technologies that have considerable promise in combating both infectious disease and cancer. However, little is known about the extrinsic factors that regulate nucleic acid vaccine responses and which may determine their effectiveness. The microbiome is recognized as a significant regulator of immune development and response, whose role in regulating some traditional vaccine platforms has recently been discovered. Using germ-free and specific pathogen-free mouse models in combination with different protein, DNA, and mRNA vaccine regimens, we demonstrate that the microbiome is a significant regulator of nucleic acid vaccine immunogenicity. Although the presence of the microbiome enhances CD8+ T cell responses to mRNA lipid nanoparticle immunization, the microbiome suppresses Ig and CD4+ T cell responses to DNA-prime, DNA-protein-boost immunization, indicating contrasting roles for the microbiome in the regulation of these different nucleic acid vaccine platforms. In the case of mRNA lipid nanoparticle vaccination, germ-free mice display reduced dendritic cell/macrophage activation that may underlie the deficient vaccine response. Our study identifies the microbiome as a relevant determinant of nucleic acid vaccine response with implications for continued therapeutic development and deployment of these vaccines.

Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke.

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

The Constitutional Isomerism of One-Component Ionizable Amphiphilic Janus Dendrimers Orchestrates the Total and Targeted Activities of mRNA Delivery.

Constitutional isomerism has been previously demonstrated by one of our laboratories to represent a powerful design strategy for the elaboration of complex functional self-organizations. Here we report the design, synthesis, and characterization of 14 positional, skeletal, and functional constitutional isomeric one-component, multifunctional, sequence-defined, amphiphilic ionizable Janus dendrimers (IAJDs). Their coassembly by simple injection with luciferase mRNA (Luc-mRNA) to form dendrimersome nanoparticles (DNPs) was studied. Subsequently, the resulting DNPs were employed to investigate, with screening experiments, the delivery of Luc-mRNA in vivo. Constitutional isomerism was shown to produce changes of up to two orders of magnitude of the total-body luciferase activity and targeted luciferase activity to the spleen and liver, of up to three orders of magnitude difference in targeted luciferase activity to the lungs and up to six orders of magnitude to lymph nodes. These results indicate that constitutional isomerism may represent not only a simple but also an important synthetic strategy that most probably may impact the activity of all components of synthetic vectors used in RNA-based nanomedicine, including in mRNA vaccines and therapeutics.

In Vivo mRNA CAR T Cell Engineering via Targeted Ionizable Lipid Nanoparticles with Extrahepatic Tropism.

With six therapies approved by the Food and Drug Association, chimeric antigen receptor (CAR) T cells have reshaped cancer immunotherapy. However, these therapies rely on ex vivo viral transduction to induce permanent CAR expression in T cells, which contributes to high production costs and long-term side effects. Thus, this work aims to develop an in vivo CAR T cell engineering platform to streamline production while using mRNA to induce transient, tunable CAR expression. Specifically, an ionizable lipid nanoparticle (LNP) is utilized as these platforms have demonstrated clinical success in nucleic acid delivery. Though LNPs often accumulate in the liver, the LNP platform used here achieves extrahepatic transfection with enhanced delivery to the spleen, and it is further modified via antibody conjugation (Ab-LNPs) to target pan-T cell markers. The in vivo evaluation of these Ab-LNPs confirms that targeting is necessary for potent T cell transfection. When using these Ab-LNPs for the delivery of CAR mRNA, antibody and dose-dependent CAR expression and cytokine release are observed along with B cell depletion of up to 90%. In all, this work conjugates antibodies to LNPs with extrahepatic tropism, evaluates pan-T cell markers, and develops Ab-LNPs capable of generating functional CAR T cells in vivo.

Nucleoside-Modified mRNA-Based Influenza Vaccines Circumvent Problems Associated with H3N2 Vaccine Strain Egg Adaptation.

Most human influenza vaccine antigens are produced in fertilized chicken eggs. Recent H3N2 egg-based vaccine antigens have limited effectiveness, partially due to egg-adaptive substitutions that alter the antigenicity of the hemagglutinin (HA) protein. The nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP) vaccine platform is a promising alternative for egg-based influenza vaccines because mRNA-LNP-derived antigens are not subject to adaptive pressures that arise during the production of antigens in chicken eggs. Here, we compared H3N2-specific antibody responses in mice vaccinated with either 3c.2A H3-encoding mRNA-LNP or a conventional egg-based Fluzone vaccine (which included an egg-adapted 3c.2A antigen) supplemented with an MF59-like adjuvant. We tested mRNA-LNP encoding wild-type and egg-adapted H3 antigens. We found that mRNA-LNP encoding wild-type H3 elicited antibodies that neutralized the wild-type 3c.2A H3N2 virus more effectively than antibodies elicited by mRNA-LNP encoding egg-adapted H3 or the egg-based Fluzone vaccine. mRNA-LNP expressing either wild-type or egg-adapted H3 protected mice against infection with the wild-type 3c2.A H3N2, whereas the egg-based Fluzone vaccine did not. We found that both mRNA-LNP vaccines elicited high levels of group 2 HA stalk-reactive antibodies, which likely contributed to protection in vivo. Our studies indicate that nucleoside-modified mRNA-LNP-based vaccines can circumvent problems associated with egg adaptations with recent 3c2.A H3N2 viruses. IMPORTANCE This study shows that the nucleoside-modified mRNA-LNP vaccine platform is a promising alternative for egg-based influenza vaccines. We show that mRNA-LNP vaccines expressing H3 antigens elicit high levels of antibodies in mice and protect against H3N2 influenza virus infection.

Development of an mRNA-lipid nanoparticle vaccine against Lyme disease.

Lyme disease is the most common vector-borne infectious disease in the United States, in part because a vaccine against it is not currently available for humans. We propose utilizing the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform to generate a Lyme disease vaccine like the successful clinical vaccines against SARS-CoV-2. Of the antigens expressed by Borrelia burgdorferi, the causative agent of Lyme disease, outer surface protein A (OspA) is the most promising candidate for vaccine development. We have designed and synthesized an OspA-encoding mRNA-LNP vaccine and compared its immunogenicity and protective efficacy to an alum-adjuvanted OspA protein subunit vaccine. OspA mRNA-LNP induced superior humoral and cell-mediated immune responses in mice after a single immunization. These potent immune responses resulted in protection against bacterial infection. Our study demonstrates that highly efficient mRNA vaccines can be developed against bacterial targets.

Targeted and Equally Distributed Delivery of mRNA to Organs with Pentaerythritol-Based One-Component Ionizable Amphiphilic Janus Dendrimers.

Delivery of nucleic acids with viral and synthetic vectors has pioneered genetic nanomedicine. Four-component lipid nanoparticles (LNPs) consisting of ionizable lipids, phospholipids, cholesterol, and PEG-conjugated lipids, assembled by microfluidic or T-tube, are the benchmark synthetic vector for delivery of mRNA. One-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) delivery systems for mRNA were developed by us to complement LNPs. IAJDs consist of multifunctional hydrophilic low-generation dendrons or minidendrons conjugated to hydrophobic dendrons. They were inspired by amphiphilic Janus dendrimers and glycodendrimers. IAJDs coassemble with mRNA into predictable-size vesicles, named dendrimersome nanoparticles (DNPs), by simple injection in acetate buffer, rather than by the complex technology required by LNPs. Assembly of DNPs by simple injection together with sequence design in the hydrophilic and hydrophobic modules of IAJDs endowed rapid screening to access discovery. Molecular design principles for targeted delivery were elaborated when the branching points of IAJDs were constructed from symmetrically and nonsymmetrically substituted plant phenolic acids interconnected by pentaerythritol (PE). Here, we report the first library containing simplified IAJDs constructed in only three steps from symmetrically trialkylated PE in the hydrophobic domain and four different piperazine-based ionizable amines in the hydrophilic part. Rapid coassembly with mRNA and in vivo screening led to the discovery of the two most active IAJDs targeting the spleen, liver, and lymph nodes, one predominantly to the spleen and liver and six delivering equally to the spleen, liver, lung, and lymph nodes. These IAJDs represent the simplest synthetic vectors and the first viral or synthetic system delivering equally to multiple organs.