Enhanced B cell priming induces broadly neutralizing HIV-1 apex antibodies

Abstract: Efficient priming of B cell precursors is a rate-limiting step in the induction of V2 apex broadly neutralizing antibodies (bNAbs)1,2. Here, we describe a novel germline-targeted HIV-1 Env (CAP256.OPT4) that increases the efficiency of V2 apex bNAb precursor priming by 30-400 fold compared with wild-type HIV-1 Envs and induces - in >90% of macaques - neutralization breadth that includes N130-containing viruses. Using three different delivery platforms - persistently replicating simian human immunodeficiency viruses (SHIVs), protein nanoparticles, and mRNA - we show bNAb priming as early as 4 weeks post-infection or immunization, and neutralization breadth in plasma by 12 weeks. In 14 SHIV-infected macaques, neutralization breadth reached as high as 90% on a 21-virus panel with potency as great as 1:20,000 (50% inhibitory dilution, ID50). Monoclonal bNAbs isolated from these animals were similarly broad and potent, with cryo-EM structures representing three distinct lineages revealing canonical needle-like HCDR3 binding. Env-Ab coevolution and structural analyses identified five key residues and loop features under positive selection and temporally associated with neutralization breadth. Importantly, prime-boost immunogens designed to capture these features induced broad and potent neutralization of globally diverse viruses including those containing N130 glycan. Further, rhesus bNAbs were not restricted to IGHD3-15*01 heavy chain alleles. These results expand the utility of the rhesus model for HIV-1 vaccine design and provide a molecular blueprint for inducing V2 apex bNAbs in rhesus and humans.

Marchitto L
Wagh K
Roark RS
Coleon S
Li H
Skelly AN
Hogarty MP
Habib R
Ding W
Ayyanathan K
Liu W
Sheng Z
Guo Y
Bal J
Smith LM
Sutherland LL
Park Y
Connell AJ
Bibollet-Ruche F
Lewis E
Plante SJ
Akeley MJ
Lora J
Zhao C
Carey JW
Martella CL
Li Y
Campion MS
Lituchy MG
Osbaldeston RA
Gordon CG
Albertus A
Su J
Noguchi C
Tam YK
Barbosa C
Liang B
Amereh K
Li X
Walsh AA
Irvine DJ
Andrabi R
Edwards RJ
Kreider EF
Weissman D
Shapiro L
Kwong PD
Korber BT
Haynes BF
Saunders KO
Hahn BH
Shaw GM


IgA is necessary and sufficient to prevent norovirus infection in mice

Abstract: Human norovirus is the leading cause of viral gastroenteritis, yet effective vaccines and therapeutics remain elusive. Using murine norovirus as a model, we found that mucosal immunoglobulin A (IgA) is both necessary and sufficient for protection against infection, whereas CD8+ T cells are dispensable. Robust intestinal IgA production requires at least 4 weeks of enteric infection, consistent with kinetics of human norovirus RNA clearance. Systemic vaccination elicits high titers of neutralizing serum IgG but fails to prevent enteric norovirus infection, phenocopying a recent human norovirus vaccine failure. In contrast, prophylactic delivery of dimeric anti-norovirus IgA via mRNA lipid nanoparticles confers sterilizing immunity. Together, these findings define a critical role for mucosal IgA in norovirus protection and identify IgA-based treatments as a therapeutic approach for human norovirus.

Ökten AB
Filler RB
Kung JL
Fang Z
McWilliams BC
Muscat-Rivera JE
Kegel MS
Olivi Gomes IM
Mankowski MC
Skelly AN
Melamed JR
Nice TJ
Lee S
Baldridge MT
Smith TJ
Wobus CE
Eisenbarth SC
Williams A
Serebryannyy LA
Douek DC
Weissman D
Chen S
Kreider EF
Craft JE
Wilen CB

Animals
Norovirus
Caliciviridae Infections
Immunoglobulin A
Mice
Humans
CD8-Positive T-Lymphocytes
Mice, Inbred C57BL
Antibodies, Viral
Immunoglobulin G


Decoupling Physisorption from Chemisorption in Clickable Lipid Nanoparticles

Abstract: Antibody conjugation is essential for targeted lipid nanoparticle (LNP) delivery, but here we show that click-chemistry produces artifacts that confound accurate measurement of covalent antibody-LNP bonding. We demonstrate that hydrophobic interactions between the most common click alkyne linker, dibenzocyclooctyne (DBCO), and the inherently hydrophobic LNP surface drive extensive nonspecific antibody physisorption, even in the absence of LNP azide groups. This physisorption yields artificially high apparent conjugation efficiencies measured by chromatographic methods. In contrast, less hydrophobic liposomes exhibit azide-dependent conjugation, highlighting a consequence of nanoparticle surface chemistry. Plasma incubation rapidly displaces physisorbed antibodies from LNPs, confirming their weak, noncovalent association, whereas covalently bound antibodies remain attached and enable effective in vivo targeting. Substituting DBCO with the less hydrophobic bicyclononyne (BCN) also reduces nonspecific associations. Our findings reveal hydrophobicity as a hidden variable in antibody-LNP conjugation and establish new standards for quantitative and reproducible measurement of targeted LNPs.

Brysgel TV
Hood ED
Milosavljevic A
Marzolini N
Zamora ME
Wu J
Shuvaev VV
Shuvaeva T
Kegel M
Muscat-Rivera J
Weissman D
Melamed JR
Myerson JW
Brenner JS
Muzykantov VR
Nong J


Preventive and therapeutic efficacy of mRNA/LNP vaccines encoding HPV16 E6 and E7 in an early-intervention HPV tumor mouse models

Abstract: Persistent infection with high-risk human papillomavirus (HPV) drives the development of cervical and other anogenital cancers, with limited therapeutic options for early-stage disease and precancerous lesions. There is a critical unmet need for effective therapeutic vaccines capable of inducing durable immune responses to eliminate early tumor cells and prevent disease progression. In this study, we developed lipid nanoparticle (LNP)-formulated mRNA vaccines encoding HPV16 E6 and E7 oncoproteins and evaluated their efficacy in a murine model using a low tumor inoculum (5,000-10,000 TC-1 cells) to represent a low tumor burden, early-intervention setting. E7 mRNA/LNP induced stronger antigen-specific T cell responses than E6 when administered intramuscularly at a low dose (1 µg). In therapeutic settings, initiated 3 d after tumor implantation, either as a single 10 µg dose or a two-dose regimen (10 µg followed by 5 µg, 6 d apart), E7 vaccination elicited robust T cell responses, significant tumor regression, and undetectable HPV16 E6/E7 DNA, outperforming E6 across regimens. In the preventive setting, administration of two 5 µg doses of HPV16 E7 prevented tumor establishment following TC-1 cell inoculation. Importantly, E7 vaccination induced durable immune memory, as demonstrated by sustained tumor protection and undetectable HPV E6/E7 DNA upon rechallenge at 5 months. These findings highlight the potential of HPV16 E7 mRNA/LNP as a therapeutic vaccine targeting low tumor burden HPV-associated disease and support further clinical development. A multivalent mRNA vaccine targeting multiple high-risk HPV genotypes is currently under development.

Saithong S
Prompetchara E
Ketloy C
Khawsang C
Tharakhet K
Kaewpang P
Yostrerat N
Wangsoontorn P
Charsangbong K
Lam K
Heyes J
Weissman D
Ruxrungtham K

Animals
Oncogene Proteins, Viral
Papillomavirus E7 Proteins
Repressor Proteins
Female
Papillomavirus Vaccines
Disease Models, Animal
Papillomavirus Infections
Mice
Vaccines, Synthetic
Nanoparticles
RNA, Messenger
T-Lymphocytes
Nanovaccines
mRNA Vaccines
Mice, Inbred C57BL
Humans
Cancer Vaccines
Liposomes


Bioinspired oxidized mRNA lipid nanoparticles for ex vivo engineering of chimeric antigen receptor macrophages targeting solid tumors

Abstract: Solid tumors remain difficult to treat via conventional and novel therapeutic strategies. Immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy have been remarkably effective in treating hematological cancers, but their efficacy is limited in solid tumors. Recently, CAR macrophages (CAR-Ms) have emerged as a promising solid tumor immunotherapy, primarily for their intrinsic tumor infiltration and effector functions. However, CAR-Ms are engineered using viral transduction, which is associated with aberrant immunogenicity and toxicity. To overcome these challenges, we developed a bioinspired oxidized lipid nanoparticle (LNP) platform for mRNA-based engineering of human CAR-Ms. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for delivery to human macrophages. The top LNP was subsequently optimized using an orthogonal design of experiments and the physicochemical properties, such as size and mRNA encapsulation, were tuned via optimization of microfluidic mixing parameters, yielding an LNP formulation that significantly outperformed a gold standard C12-200 LNP. Utilizing small molecule and antibody inhibitors, we demonstrate that uptake of optimized LNPs into macrophages is driven by apolipoprotein E independent macropinocytosis, which is further supported by potent extrahepatic spleen tropism upon intravenous administration to mice. Lastly, we demonstrate the translatability of this LNP platform and utilize it to engineer functional primary human HER2-CAR-Ms ex vivo with potent antigen-specific tumor cell killing, validated in an ex vivo co-culture with ovarian cancer cells. This bioinspired oxidized LNP platform demonstrates potential for engineering a range of human CAR-M immunotherapies to treat various types of solid tumors.

Mukalel AJ
Tylek T
Geisler HC
O'Brien E
Frazee C
Li J
Thatte AS
Safford HC
Figueroa-Espada CG
Alameh MG
Hamilton AG
Mai D
Sheppard NC
June CH
Weissman D
Spiller KL
Mitchell MJ


Antigen-specific messenger RNA lipid nanoparticle therapy with mTOR inhibition promotes regulatory T cells and limits allergy

Abstract: Immune tolerance in allergic diseases is associated with attenuation of TH2 responses by shifting of antigen-specific immunity toward TH1 and regulatory T-cell pathways, but current strategies incompletely induce durable regulatory immunity. We determined whether combining an allergen-encoded messenger RNA (mRNA) lipid nanoparticle (LNP) vaccine with inhibition of the mechanistic target of rapamycin (mTOR) enhances regulatory T-cell responses. Mice were immunized with an allergen-encoded mRNA-LNP vaccine alone or in combination with an mTOR inhibitor, followed by induction of a preclinical model of allergic asthma. Antigen-specific T-cell responses, eosinophil activation, airway hyperresponsiveness, mucus production, and markers of cytotoxicity were assessed. Immunization with allergen-encoded mRNA-LNP elicited TH1-associated and cytotoxic CD8+ T responses that counterbalanced TH2 immunity. Coadministration with an mTOR inhibitor shifted this profile by promoting generation of functional regulatory T cells and attenuating IFN-γ production and CD8+ T-cell responses. This combinatorial strategy preserved the antiallergic effects of mRNA-LNP immunization, reduced eosinophil activation markers, and limited vaccine-associated cytotoxicity. The ability of an mTOR inhibitor to profoundly modify mRNA-LNP therapy by inducing regulatory T cells presents a potential strategy to enhance regulatory immunity in the treatment of allergy and other inflammatory diseases.

The Journal of allergy and clinical immunology

Rochman Y
Melamed JR
Klingler AM
Kotliar M
Rochman M
Caldwell JM
Felton JM
Osswald GA
Muscat-Rivera J
Kegel M
Barski A
Lewkowich IP
Weissman D
Rothenberg ME


mRNA delivery of a class 1/4 SARS-CoV-2 neutralizing antibody protects against diverse sarbecoviruses in a lethal mouse challenge model

Abstract: The unyielding antigenic drift of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as the threat of future zoonotic sarbecovirus spillovers, has prompted the search for broadly neutralizing antibodies (bNAbs) to inform rational therapeutic and vaccine design. Here, we isolated and characterized 20 receptor binding domain (RBD)-directed bNAb lineages from a serially sampled SARS-CoV-2 patient who was infected and vaccinated during the early months of the pandemic. Thirteen of these targeted the highly conserved, cryptic class 1/4 or class 4 RBD epitopes and had long (18 to 26 amino acid) heavy chain complementarity determining region 3 loops that utilized the IGHD3-22 gene segment. Five bNAbs potently neutralized all 18 viruses in a panel containing SARS-CoV-2 variants up to the recently emerged XBB.1.5 and JN.1 strains as well as diverse sarbecoviruses from other clades. Structural analyses of the Ab401 and Ab568 bNAbs complexed with RBD and Spike trimer, respectively, revealed recognition features in common with other class 1/4 bNAbs. Prophylactic administration of Ab401 as a recombinant protein afforded robust protection against infectious challenge with either SARS-CoV-2_WA1 or a related bat sarbecovirus with zoonotic potential. A similar level of protection was achieved when the heavy and light chains of Ab401 were delivered as lipid nanoparticle-encapsulated mRNAs. These data expand the arsenal of SARS-CoV-2 bNAbs for clinical development and identify mRNA-based antibody delivery as a promising platform for both pandemic preparedness and protection of immunocompromised patients against emerging sarbecovirus variants.

Proceedings of the National Academy of Sciences of the United States of America

Skelly AN
Fan C
Keeffe JR
Ökten AB
Gavor E
Newby ML
Allen JD
Kreider EF
Ding W
Osbaldeston RA
Park Y
Connell AJ
Lituchy MG
Bibollet-Ruche F
Radford KM
P West A Jr
Peña-Hernández MA
Cruickshank K
Liu W
Li Y
Albertus A
McWilliams B
Russell RM
Konrath KM
Torres JL
Yuan M
Gao H
Montefiori DC
Saag MS
Goepfert PA
Kulp DW
Ward AB
Wilson IA
Shaw GM
Andrabi R
Crispin M
Weissman D
Wilen CB
Bjorkman PJ
Hahn BH

Animals
SARS-CoV-2
COVID-19
Mice
Antibodies, Viral
Antibodies, Neutralizing
Humans
Epitopes
Spike Glycoprotein, Coronavirus
RNA, Messenger
Disease Models, Animal


Plug-and-play assembly of biodegradable ionizable lipids for potent mRNA delivery and gene editing in vivo

Abstract: mRNA-based gene editing therapeutics offer the potential to permanently cure diseases but are hindered by suboptimal delivery platforms. Here, we devise a robust combinatorial chemistry for the plug-and-play assembly of structurally diverse biodegradable ionizable lipids from amines/thiols and dialkyl maleates. After screening 500 ionizable lipids, we obtained structure-activity relationships essential for effective in vitro mRNA delivery with the help of machine learning. Furthermore, we identified a lead ionizable lipid candidate that produced potent lipid nanoparticles for the delivery of various gene editing tools in wild-type and genetically modified mice compared to literature and industry benchmark lipid nanoparticles. Mechanistically, our lipid nanoparticles show favorable physicochemical properties, which could synergistically contribute to the superior delivery performance. This study highlights the utility of this synthetic method as well as the generality of this platform for potent in vivo gene editing.

Han X
Xu Y
Ricciardi AS
Xu J
Xiang Y
Palanki R
Chowdhary V
Xue L
Gong N
Alameh MG
Peranteau WH
Wilson JM
Reker D
Weissman D
Mitchell MJ

Animals
Lipids
RNA, Messenger
Gene Editing
Mice
Nanoparticles
Gene Transfer Techniques
Humans
Structure-Activity Relationship
Liposomes


In vivo reprogramming of cytotoxic effector CD8 T cells via fractalkine-conjugated mRNA-LNPs

Abstract: Selective in vivo reprogramming of cytotoxic effector CD8 T (Teff) cells holds tremendous promise as a therapeutic tool but has not yet been accomplished. Here, we demonstrate that fractalkine-conjugated mRNA lipid nanoparticles (mRNA-LNPs) can specifically target and deliver mRNA to CX3CR1+ Teff cells in vitro and in vivo. In mice, fractalkine-conjugated mRNA-LNPs targeted up to 95% of blood and splenic Teff cells. In addition, delivery of IL-2-encoding mRNA and human CD62L-encoding mRNA to mouse Teff cells enabled robust exogenous IL-2 secretion and CD62L expression. In rhesus macaques, fractalkine-conjugated mRNA-LNPs targeted up to ~100% of peripheral blood Teff cells, and delivery of human CD62L-encoding mRNA enabled cell-surface human CD62L expression on peripheral blood Teff cells and detection of human CD62L+ Teff cells in lymphoid tissue. Collectively, these data demonstrate the potential of natural receptor ligand-based targeting of mRNA-LNPs for rapid, efficient, and transient in vivo modification of Teff cells.

Corrigan AR
Ngiow SF
Statzu M
Albertus A
Pampena MB
Nordin JML
Carro SD
Harper J
Stammen RL
Wood J
Ni H
Su J
Hajialyani M
Shuvaev VV
Alcalde V
Ali MA
Hamilton JT
Ramalingam R
Wu VH
Paiardini M
Weissman D
Wherry EJ
Kreider EF
Betts MR

Animals
RNA, Messenger
Humans
Mice
Chemokine CX3CL1
Macaca mulatta
Nanoparticles
L-Selectin
T-Lymphocytes, Cytotoxic
Mice, Inbred C57BL
CD8-Positive T-Lymphocytes
Liposomes


Induction of broadly neutralizing HIV antibodies by a two-step mechanism informs vaccine design

Abstract: A major obstacle confronting HIV-1 vaccine and cure research is the lack of an outbred animal model for rapid and consistent induction of broadly neutralizing antibodies (bNAbs). We designed an epitope-focused simian-human immunodeficiency virus (SHIV.5MUT) that elicited broad and potent V3-glycan-targeted antibodies within a year of infection in 14 of 22 macaques compared with 0 of 14 control animals. SHIV.5MUT elicited bNAbs by a two-step mechanism, inducing an initial wave of V1-directed antibodies that selected for envelope (Env) glycoprotein variants with shortened, hypoglycosylated V1 loops, which in turn primed V3-glycan bNAb precursors. Rhesus bNAbs were immunogenetically and structurally diverse, closely resembling human V3-glycan bNAbs. Env-bNAb coevolution revealed a diverse repertoire of bNAb precursors and the Env variants that matured them, yielding a molecular blueprint for vaccine design.

Skelly AN
Gristick HB
Li H
Gavor E
Connell AJ
Kreider EF
Marchitto L
Hogarty MP
Newby ML
Allen JD
Liu W
West AP Jr
Ayyanathan K
Campion MS
Winters K
Gordon CG
Osbaldeston RA
Akeley MJ
Lewis E
Li Y
Singh A
Cruickshank K
Park Y
Zhao C
Li X
Amereh K
Van Itallie E
Carey JW
Albertus A
DeLaitsch AT
Keeffe JR
Lituchy MG
Walsh AA
Morris DJ
Habib R
Bibollet-Ruche F
Mishra N
Avillion G
Koranda NS
Plante SJ
Martella CL
Lora J
Wang EJD
Lewis MG
Martin MA
Nussenzweig MC
Seaman MS
Irvine DJ
Wiehe KJ
Haynes BF
Wagh K
Korber B
Andrabi R
Crispin M
Weissman D
Bjorkman PJ
Hahn BH
Shaw GM

env Gene Products, Human Immunodeficiency Virus


Effect of Acid-Stabilizing Hemagglutinin Mutations on Immunogenicity and Heterologous Protection by H1N1 Influenza Virus mRNA-LNP Vaccines

Abstract: While current influenza vaccines often lack broad protection against antigenically drifted strains, some modified hemagglutinin (HA) protein antigens have shown promise in eliciting broadly neutralizing antibodies against conserved epitopes. During infection, the mildly acidic environment of the late endosome triggers irreversible HA conformational changes resulting in a post-fusion structure with altered antigenicity. While enhancing the stability of other structural class I viral fusion protein antigens has been instrumental in improving the effectiveness of COVID-19 and RSV vaccines, the role of HA stability in influenza vaccine immunogenicity is relatively unclear. Here, we used the nucleoside-modified mRNA-LNP platform to test engineered HA antigens with specific acid-stabilizing mutations (E47K, K58I, R106K, and K153E) in the HA stalk. All mutations increased HA acid stability, but E47K and R106K did not increase immunogenicity. K153E and K58I, but not E47K and R106K, enhanced the cell-surface expression of the HA protein in vitro. In mice, K153E- and K58I-containing mRNA-LNP vaccines elicited increased neutralizing antibody titers against homologous virus. K153E conferred greater protection than wild-type vaccine against lethal heterologous A/PR/8/34 challenge at low doses (0.5-1.0 µg), despite the absence of neutralizing antibodies against the challenge strain. K153E also elicited greater expansion of antigen-specific antibody-secreting cells (ASCs) in the bone marrow, as well as cross-reactive T follicular helper (Tfh) cells in the spleen. For the vaccines studied, increased HA expression was a stronger correlate of mRNA-LNP enhancement than increased HA stability.

Ojha CR
Rovito SW
Banoth B
Kim H
Jones JC
Alameh MG
Chen PL
Webby RJ
Weissman D
Russell CJ

Hemagglutinin Glycoproteins, Influenza Virus


Investigating the impact of hypoxia and syncytialization on lipid nanoparticle-mediated mRNA delivery to placental cells

Abstract: Placental dysfunction leads to pregnancy-related disorders that affect up to 15% of pregnancies. Several of these, such as preeclampsia, are symptomatically managed but have no curative treatments other than preterm delivery. Placental dysfunction arises from improper placental development, leading to restricted blood vessel formation and a hypoxic placental microenvironment. The development of placental therapeutics is challenging due to the complex physiology that enables the placenta to control uptake and transport. Here, we use a simple culture system that combines hypoxia and trophoblast syncytialization to model the functional syncytiotrophoblast layer of the placenta under hypoxic stress. Using this model, we evaluate the impact of hypoxia on lipid nanoparticle (LNP)-mediated mRNA delivery. Our data show that hypoxia hinders syncytiotrophoblast formation in vitro. Despite this, LNP delivery to syncytiotrophoblasts increases protein translation and secretion, particularly under hypoxic conditions. Further, we show delivery of a therapeutic mRNA, placental growth factor (PlGF), to syncytiotrophoblasts in hypoxia, which restored diminished PlGF levels back to normoxic controls. These findings provide an LNP platform for efficient mRNA delivery to hypoxic trophoblasts and demonstrate the importance of considering hypoxia towards the development of drug delivery platforms for placental therapeutics.

Young RE
Vijayakumar T
Reilley LJ
Darji K
Patel D
Hofbauer S
Alameh MG
Weissman D
Riley R


Combining CRISPR/Cas9-mediated TRAC knockout with mRNA-based CAR expression enables flexible generation of allogeneic CAR T cells

Abstract: Autologous chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable efficacy in hematologic malignancies but remain limited by complex manufacturing processes. Allogeneic, off-the-shelf CAR T cells derived from healthy donors represent a promising alternative; however, safe implementation requires elimination of endogenous T-cell receptor (TCR) expression and flexible CAR expression strategies. This study aimed to develop an optimized manufacturing workflow for allogeneic CAR T cells by combining CRISPR/Cas9-mediated TCR knockout with mRNA-based CAR expression, and to evaluate cryopreservation strategies enabling on-demand CAR T-cell generation. Healthy donor T cells were edited at the TRAC locus using CRISPR/Cas9 to generate TCR-deficient T cells. These cells were cryopreserved and subsequently transfected with mRNA encoding CD117, BCMA, or CD19 CARs. CAR expression, cell viability, immunophenotype, cytokine secretion, and antigen-specific cytotoxicity were assessed under different cryopreservation-transfection conditions. TCR knockout T cells exhibited efficient TCR disruption with reduced alloreactive proliferation. CD117 mRNA CAR T cells derived from TCR-deficient T cells demonstrated CAR expression kinetics, immunophenotypic profiles, and antigen-specific cytotoxicity comparable to wild-type CAR T cells. Evaluation of two cryopreservation strategies revealed that cryopreservation prior to mRNA electroporation preserved cell viability, phenotype, and cytotoxic function, whereas cryopreservation after mRNA transfection was associated with reduced functional activity. The optimized protocol was successfully extended to CD19- and BCMA-targeting CAR mRNAs. Collectively, these findings establish a modular platform for producing allogeneic CAR T cells using mRNA technology, offering a practical approach for rapid, on-demand CAR T-cell therapy.

Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Buakaew T
Thaiwong R
Inthanachai T
Palaga T
Weissman D
Suppipat K
Ausavarungnirun C
Tawinwung S

Humans
RNA, Messenger
CRISPR-Cas Systems
T-Lymphocytes
Cryopreservation
Receptors, Chimeric Antigen
Immunotherapy, Adoptive
Receptors, Antigen, T-Cell
Cell Survival
Gene Knockout Techniques
Transfection


One-component ionizable amphiphilic Janus dendrimers as a delivery platform for efficient mRNA vaccine development

Abstract: There have been rapid advances in nucleoside-modified messenger RNA-based vaccines, particularly during the SARS-CoV-2 pandemic, demonstrating a rapid and cost-effective approach for addressing infectious diseases. A major challenge remains in developing a delivery system that protects mRNA from degradation and facilitates its passage through tissue and cellular barriers. Current four-component lipid nanoparticles enable efficient endosomal escape and are a pivotal technology for the delivery of mRNA vaccines. However, challenges such as stability, availability, durability, and adverse effects persist. We have developed a self-assembling one-component ionizable amphiphilic Janus dendrimers (IAJD) delivery system with naturally occurring amino heads, capable of efficiently delivering mRNA to the spleen and lymph nodes. This single-component system simplifies synthesis, reduces development complexity, and enables rapid global distribution of mRNA vaccines during pandemics. As a proof of concept, one-component IAJD97, formulated with mRNA encoding norovirus mRNA capsid protein, demonstrates the potential of IAJDs as efficient delivery platform for mRNA vaccines, advancing their effectiveness and expanding applications to improve public health outcomes.

Ona NA
Zhang D
Lindesmith LC
Park WJ
Meshanni JA
Berkihiser S
Vasserman JA
Toshtzar S
Moore NC
Williams B
Nakagawa B
Baboo I
Pan L
Cheng W
Mercado-López X
Bonilla-Acosta W
Brewer-Jensen PD
Zweigart MR
Reyes YI
Mallory ML
May SR
Zhou Y
Percec V
Baric RS
Li Y
Weissman D
Atochina-Vasserman EN

Dendrimers
Animals
RNA, Messenger
Humans
SARS-CoV-2
COVID-19 Vaccines
COVID-19
Vaccine Development
mRNA Vaccines
Nanoparticles
Mice


Liver-Detargeted Aromatic Bioreducible mRNA Lipid Nanoparticles Confer Lymph Node Tropism and Robust Antigen-Specific Immunity

Abstract: Lipid nanoparticles (LNPs) have been immensely successful in facilitating the delivery of nucleic acids to tissues of interest and continue to be optimized for mRNA delivery. These vehicles are particularly advantageous for vaccination due to their ease of production, highly tunable composition, and ability to induce robust immune responses without an adjuvant, as exemplified most recently by the clinical success of the Pfizer/BioNTech and Moderna COVID-19 vaccines. However, LNPs are known to exhibit off-target liver tropism. To address this limitation, we developed a library of aromatic bioreducible ionizable lipids that potently transfect secondary lymphoid tissues with liver detargeting capabilities compared with an industry standard ionizable lipid used in the COVID-19 vaccine. The library consists of three modular components: amine core structure, lipid tail length, and regiochemistry. These aromatic ionizable lipids employ benzene rings both as a scaffold for regiochemical differences and as a moiety to improve transfection. Bioreducible disulfide bonds in the lipids additionally serve to increase their biodegradability. When these aromatic ionizable lipids are formulated as LNPs, top-performing aromatic LNPs (aroLNPs) accumulate in and transfect lymph nodes while minimizing off-target liver tropism. Top-performing aroLNPs also induce strong antigen-specific immune responses, increased effector memory T cell generation, and decreased terminal effector T cell generation in mice when utilized in a preclinical SARS-CoV-2 vaccine study. Additionally, aroLNPs are strongly retained in the injection site and induce low levels of systemic inflammatory cytokines. Together, these results establish aroLNPs as a promising platform for vaccine delivery and potentially other immune-focused therapeutic applications.

Journal of the American Chemical Society

Yamagata HM
Padilla MS
Hamilton AG
Swingle KL
Thatte AS
Ricciardi AS
Agrawal A
Fitzgerald E
Poirier AJ
Geisler HC
Joseph RA
Chalom OZ
Du S
Li JJ
Kim D
Whitaker RC
Figueroa-Espada CG
Han EL
Murray AM
Alameh MG
Weissman D
Mitchell MJ

Animals
Nanoparticles
Lipids
Mice
Liver
Lymph Nodes
RNA, Messenger
COVID-19 Vaccines
Humans
SARS-CoV-2
COVID-19
Mice, Inbred C57BL
Liposomes

Journal Article
Research Support, Non-U.S. Gov't


Preventing Disease Emergence Following Eradication: Application to Mpox

Abstract: We use simple mathematical models to explore the factors that influence the evolutionary emergence of mpox to a pathogen capable of sustained human to human transmission that poses a global threat. Smallpox eradication followed by the discontinuation of immunization with vaccinia has led to a decline in the level of population immunity against related poxviruses such as mpox. This decline in immunity results in an increase in both the number of spillovers and the extent of human to human transmission. We find that increases in transmissibility of mpox between humans have a much greater effect on the probability of evolutionary emergence compared with increases in the number of zoonotic spillovers. We suggest that while mpox only needs to have a reproductive number slightly greater than one to become endemic, subsequent adaptation is likely to further increase its transmissibility in the human population. As a consequence a much higher level of vaccination (or other intervention) is needed to control the pathogen after its evolutionary emergence compared with what is needed to prevent it from emerging in the first place.

bioRxiv : the preprint server for biology

Hirst C
Deichmann J
Saha A
Longini I
Handel A
Lipsitch M
Weissman D
Antia R


Prodrug-tethered lipid nanoparticles for synergistic messenger RNA cancer immunotherapy

Abstract: Regulating T cell phenotypes between activation and exhaustion remains a significant challenge for messenger RNA-based cancer immunotherapy. A potential approach to improve anti-cancer T cell activity is to co-deliver interleukin-12 (IL-12), to stimulate effector T cells, and indoleamine 2,3-dioxygenase (IDO) inhibitor, to suppress T cell exhaustion. Here we design prodrug ionizable lipid nanoparticles (pLNPs), via a library of prodrug ionizable lipids (pILs), incorporating an intracellularly cleavable IDO inhibitor within the pIL structure and encapsulating IL-12 messenger RNA. The lead pIL shows enhanced mRNA transfection over a clinically utilized ionizable lipid, as well as strong immunomodulatory effects via release of the IDO inhibitor. In a subcutaneous colon cancer mouse model, pLNP drives complete regression of primary tumours by eliciting effector T cell infiltration while reducing exhaustion, induces a memory T cell response and stimulates a systemic immune response that allows for regression of distal tumours in this study. These results highlight the promise of pLNPs for small-molecule drug and mRNA combination cancer immunotherapy.

Shi Q
Gong N
Wang J
Palanki R
Zheng Q
Alameh MG
Dwivedi G
Davis B
Melamed J
Luo Z
Xu J
Figueroa-Espada CG
Xue L
Zeng Y
Han X
Kim D
Chen Q
Yamagata H
Geisler HC
El-Mayta R
Yoon IC
Weissman D
Mitchell MJ

Prodrugs
Animals
Nanoparticles
Immunotherapy
Mice
RNA, Messenger
Lipids
Interleukin-12
Cell Line, Tumor
Humans
Indoleamine-Pyrrole 2,3,-Dioxygenase
Mice, Inbred C57BL
Colonic Neoplasms
T-Lymphocytes
Female
Liposomes


Evaluation of an H5 influenza virus mRNA-lipid nanoparticle (LNP) vaccine in lactating dairy cows

Abstract: Highly pathogenic avian influenza (HPAI) clade 2.3.4.4b H5N1 virus has recently emerged in dairy cattle in the United States. The virus replicates primarily in the mammary gland of infected cattle, leading to dramatic reductions in milk production. It is thought that the virus transmits from animal to animal through viral shedding in milk, and therefore, vaccines that decrease the amount of virus in milk can potentially limit the current outbreak and reduce the risk of H5N1 spillover into humans. Here, we assess the immunogenicity and efficacy of a clade 2.3.4.4b H5 mRNA-LNP vaccine in lactating dairy cows. We found that the H5 mRNA-LNP vaccine elicited robust antibody responses in sera and milk and significantly reduced viral replication and disease caused by clade 2.3.4.4b H5N1 intramammary infection.

Santos JJS
Souza CK
Zanella GC
Goulart DB
Arruda B
Boggiatto P
Palmer MV
Snyder CA
Kristula MA
Dickens C
Webb TL
Atkinson RK
Dadonaite B
Dwivedi G
Alameh MG
Bloom JD
Weissman D
Althouse GC
Baker AL
Hensley SE


Crosslinked ionizable lipids reprogram dendritic cell metabolism for potent mRNA vaccination

Abstract: Modulating metabolism in immune cells is an effective approach to induce desired immune responses. Here we develop a lipid nanoparticle (LNP) capable of metabolic reprogramming of dendritic cells for mRNA vaccine applications. Using imidoester-based conjugation chemistry, we design a crosslinked ionizable lipid, C12-2aN, which possesses intrinsic metabolic modulatory properties. This multifunctional ionizable lipid not only promotes effective mRNA expression by facilitating endosomal escape but also stimulates glycolysis through mTORC2 pathway activation. As both an mRNA carrier and a metabolic modulator, C12-2aN LNPs lead to potent vaccine efficacy in both SARS-CoV-2 and OVA cancer vaccine models, resulting in stronger neutralization of pseudovirus infection and improved survival rates, respectively, compared with control LNPs without the crosslinker. Moreover, C12-2aN LNPs outperformed FDA-approved LNPs in terms of reduced off-target delivery and lower immunogenicity. Overall, the integration of mRNA delivery and metabolic reprogramming induced by the ionizable lipid component presents significant potential for next-generation mRNA LNP vaccines.

Kim D
Gong N
Alameh MG
Han EL
Wang H
Wang J
Feng E
Yoon IC
Murray AM
Shi Q
Moon SJ
Mrksich K
Weissman D
Mitchell MJ


Efficacy and immunological correlates of an mRNA-LNP vaccine for protection against an emerging rickettsial pathogen

Abstract: A rickettsial pathogen, Ehrlichia chaffeensis, causes an emerging tick-borne disease called human monocytic ehrlichiosis (HME). E. chaffeensis in ticks expresses a porin OMP-1B, the immunodominant major outer membrane protein, which is required for nutrient uptake across the outer membrane of bacteria. We developed an OMP-1B-encoding mRNA-lipid nanoparticle (hereafter OMP-1B mRNA-LNP) vaccine. Immunization of naïve mice with OMP-1B mRNA-LNP showed significant protection against E. chaffeensis infection following a challenge with E. chaffeensis-infected tick cells. Following vaccination, increased OMP-1B-specific serum IgG, IgG1, and IgG2a titres, as well as E. chaffeensis infection-neutralizing antibodies were detected. An ELISpot assay revealed significant increase in OMP-1B-specific IFN-γ-secreting cells in the blood and spleen samples from mice vaccinated with OMP-1B mRNA-LNP. Flow cytometry analysis of spleen samples showed that OMP-1B antigen-specific IFN-γ-producing CD4+ and CD8+ T cells as well as granzyme B-producing cytotoxic CD8+ T cells were significantly increased in the vaccinated mice. Our results demonstrate that an mRNA vaccine targeting OMP-1B conferred protection against E. chaffeensis infection, with significant humoral immune responses including infection-neutralizing antibodies, balanced Th1/Th2 response, and antigen-specific T helper and cytotoxic T cell activation. These data suggest that the mRNA-LNP approach is a viable strategy for developing efficient anti-rickettsial vaccines.

Lin M
Denton S
Duan N
Iqbal N
Boyaka PN
Dwivedi G
Alameh MG
Weissman D
Rikihisa Y

Animals
Mice
Antibodies, Bacterial
Female
Bacterial Outer Membrane Proteins
Immunoglobulin G
RNA, Messenger
Porins
Bacterial Vaccines
Nanoparticles
CD4-Positive T-Lymphocytes
CD8-Positive T-Lymphocytes
Interferon-gamma
Antibodies, Neutralizing
Humans
Ticks
Vaccination
Liposomes


Targeted Lipid Nanoparticle Delivery of FAP-CAR mRNA Enables Potent In Vivo T-cell Engineering against Pancreatic Tumors

Abstract: Fibroblast activation protein (FAP), which is highly expressed on cancer-associated fibroblasts (CAF), is a promising therapeutic target to achieve normalization of the tumor microenvironment. We previously established an ex vivo retroviral-transduced FAP-specific chimeric antigen receptor (FAP-CAR) T-cell approach to deplete FAP+ CAFs that resulted in delayed tumor growth associated with disruption of desmoplastic matrix and enhanced immune cell infiltration and reversed immune exclusion and immunosuppression. In this study, we describe an in vivo strategy for generating FAP-CAR T cells using anti-CD5-conjugated targeted lipid nanoparticles (tLNP) encapsulating FAP-CAR mRNA and assessed the efficacy of this approach compared with adoptive transfer of retrovirus-transduced CAR T cells in a preclinical model of pancreatic ductal adenocarcinoma. With transient CAR expression in >45% of splenic, >69% of circulating, and >35% of tumor-infiltrating T cells, the abundance of peripheral and intratumoral FAP-CAR+ T cells detected following a single intravenous dose of FAP-CAR mRNA tLNPs was greater than that detected following administration of 1 × 107ex vivo retrovirally transduced FAP-CAR T cells. Furthermore, in vivo mRNA CAR T-cell engineering resulted in as good or greater inhibition of tumor growth as compared with adoptive transfer of ex vivo retroviral-engineered T cells. Given that in vivo generation of CAR T cells resulted in transient CAR expression and circumvented the need for autologous T-cell isolation, viral vectors, and lymphodepletion, this platform represents a potentially safer, more accessible, and cost-effective method for targeting stromal cells to normalize the tumor microenvironment in desmoplastic tumors and has potential implications for tumor antigen-targeted CAR T cells.

Bajbouj K
Xiao Z
Todd L
Huang L
Papp TE
Halilovic F
Ramani J
Bao Y
Butcher M
Bot A
Aghajanian H
June CH
Weissman D
Parhiz H
Albelda SM
Puré E

Animals
Fibroblast Activation Protein Alpha
Nanoparticles
Humans
Pancreatic Neoplasms
Endopeptidases
RNA, Messenger
T-Lymphocytes
Receptors, Chimeric Antigen
Mice
Immunotherapy, Adoptive
Gelatinases
Membrane Proteins
Serine Endopeptidases
Cell Line, Tumor
Lipids
Tumor Microenvironment
Female
Xenograft Model Antitumor Assays
Liposomes


Neonatal mice immune response to COVID-19 mRNA vaccine

Abstract: A vaccine based on an mRNA platform was first licensed for human use during the COVID-19 pandemic. However, data on the immunogenicity of SARS-CoV-2 mRNA vaccines in neonates is insufficient and the vaccines were only authorized for those over six months of age. Here, we compared the antibody responses induced by both recombinant receptor binding domain (rRBD) of SARS-CoV-2 spike protein (RBD) and mRNA encoded RBD (RBD-mRNA-LNP) forms in neonatal mice. When administered twice three weeks apart, both forms induced detectable anti-RBD antibodies. However, levels of IgG antibodies against RBD were significantly higher with more potent inhibition of RBD binding to ACE2 in neonatal mice immunized with RBD-mRNA-LNP vaccine compared to those immunized with rRBD vaccine adjuvanted with AddaVax™, a squalene-based adjuvant. Thus, the mRNA vaccine platform elicits higher levels of antibodies with improved functional capability in neonatal mice compared to the recombinant protein platform.

Lotspeich-Cole L
Jha MK
Parvathaneni S
Lee RC
Weissman D
Major M
Akkoyunlu M

Animals
COVID-19 Vaccines
Mice
Antibodies, Viral
Spike Glycoprotein, Coronavirus
Animals, Newborn
COVID-19
Vaccines, Synthetic
SARS-CoV-2
Immunoglobulin G
Female
mRNA Vaccines
Adjuvants, Immunologic
RNA, Messenger
Angiotensin-Converting Enzyme 2
Mice, Inbred BALB C


High-Throughput In Vivo Screening Using Barcoded mRNA Identifies Lipid Nanoparticles With Extrahepatic Tropism for In Situ Immunoengineering

Abstract: Interest continues to grow in the use of mRNA vaccines and therapeutics. While effective for immunization against infectious diseases, lipid nanoparticle (LNP) formulations used for other mRNA delivery applications suffer from off-target accumulation, poor immune transfection, and reactogenicity, limiting their application to immunoengineering. Development of new mRNA LNPs is severely bottlenecked by the LNP discovery process, which is historically low-throughput due to reliance on low-plex measurements. Here, we develop a high-throughput in vivo mRNA LNP screening platform based on barcoded mRNA (b-mRNA). Using this b-mRNA screening platform to simultaneously evaluate 122 LNPs, we identify novel LNP formulations capable of potent hepatic and extrahepatic transfection. We evaluate a lead LNP candidate for in situ immune modulation in a syngeneic mouse model of melanoma and demonstrate a significant reduction in tumor burden and extended survival compared to mice treated with a gold standard mRNA LNP formulation. We employ novel biochemical characterization techniques to analyze nanoparticle protein corona formation with single-particle resolution and gain insight into the influence of protein adsorption on hepatic and splenic transfection. Together, our results demonstrate the value of advanced LNP screening and characterization techniques for the development of next-generation mRNA LNPs for immunoengineering.

Advanced materials (Deerfield Beach, Fla.)

Hamilton AG
Thatte AS
Xu J
Luo Z
Safford HC
Swingle KL
Muscat-Rivera J
Kegel M
Han X
Joseph RA
Murray AM
Geisler HC
Whitaker RC
Xue L
Spektor R
Melamed JR
Weissman D
Mitchell MJ

Animals
Nanoparticles
RNA, Messenger
Lipids
Mice
High-Throughput Screening Assays
Cell Line, Tumor
Transfection
Liver
Female
Liposomes


Preclinical evaluation of DENV1 mRNA vaccines in mice: Toward improved neutralizing antibody and T cell responses

Abstract: Dengue virus (DENV) infection represents a significant public health concern in tropical and subtropical areas, with increasing cases in parts of Europe. Currently, licensed vaccines, Dengvaxia® and Qdenga®, rely on live-attenuated platforms but show limited efficacy due to imbalanced immune responses and risks associated with antibody-dependent enhancement (ADE). In this study, we developed and evaluated nucleoside-modified mRNA vaccine encoding the dengue virus serotype 1 (DENV1) pre-membrane and envelope (prME) proteins. To reduce the potential for ADE, we incorporated the F108A mutation in the fusion loop, substituted the pr region with that from Japanese encephalitis virus (JEV), and incorporated a consensus envelope domain III (cEDIII) sequence to broaden neutralizing antibody responses. Immunogenicity was assessed in BALB/C mice immunized twice at three-week intervals with 1, 2.5, or 10 μg doses. The WT prME construct induced the highest DENV1 neutralizing antibody (NtAb) titers in a dose-dependent manner and robust T cell responses, particularly with the 2.5 μg dose. While the F108A and cEDIII modifications modulated antigen expression and broadened cross-reactivity, they also slightly reduced DENV-1-specific neutralizing titers. Among constructs, F108A+cEDIII demonstrated reduced ADE activity with improved cross-neutralization, especially against DENV3. However, the prJEV chimera exhibited low immunogenicity, likely due to prM-E domain incompatibility. Overall, the WT prME construct showed the most favorable balance of immunogenicity and safety, supporting its advancement as a prototype for future tetravalent dengue mRNA vaccine development.

Khawsang C
Prompetchara E
Saithong S
Tharakhet K
Kaewpang P
Yostrerat N
Wangsoontorn P
Pitakpolrat P
Buranapraditkun S
Puttikhunt C
Lam K
Heyes J
Ketloy C
Weissman D
Ruxrungtham K

Animals
Antibodies, Neutralizing
Dengue Virus
T-Lymphocytes
Mice, Inbred BALB C
Dengue Vaccines
Mice
Antibodies, Viral
Dengue
Female
RNA, Messenger
mRNA Vaccines
Viral Envelope Proteins


Targeting DNA-LNPs to Endothelial Cells Improves Expression Magnitude, Duration, and Specificity

Abstract: DNA-lipid nanoparticles (DNA-LNPs) loaded with inhibitors of the cGAS-STING pathway enable safe and effective delivery of DNA in vivo. Herein, we report the first instances of extrahepatic DNA-LNP targeting. DNA-LNPs conjugated to antibodies against PECAM-1 or VCAM-1 target the endothelium of the lungs and brain/spleen, respectively. These LNPs drive robust transgene expression in their target organs, with greater magnitude and duration than untargeted LNPs. Lung specificity of PECAM-targeted transgene expression increases over two weeks, resulting in markedly higher lung-to-liver expression ratios than our previous PECAM-targeted mRNA-LNPs. Off-target liver DNA expression declines to undetectable levels but persists in the lungs, while mRNA expression uniformly decreases due to its short half-life. We further improve this expression specificity by replacing full-length antibodies with Fab fragments. Single-cell analysis reveals a key mechanism underlying the improvements in organ-specificity: target organ expression is dominated by long-lived endothelial cells, while off-target liver delivery and expression are in non-endothelial cells with shorter half-lives. Collectively, these studies demonstrate that targeted DNA-LNPs achieve high levels of organ- and cell-type-specific transgene expression and thus provide a therapeutic platform for dozens of endothelial-centric diseases.

Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Marzolini N
Brysgel TV
Rahman RJ
Essien EO
Nwe SY
Wu J
Majumder A
Patel MN
Tiwari S
Espy CL
Dong F
Santos-De León SA
Shah A
Shuvaev VV
Hood ED
Chase LS
Weissman D
Katzen JB
Frank DB
Bennett ML
Marcos-Contreras OA
Myerson JW
Muzykantov VR
Reyes-Esteves S
Brenner JS

Platelet Endothelial Cell Adhesion Molecule-1


An influenza HA mRNA-LNP vaccine induces potent responses in newborn nonhuman primates that enhance protection from challenge

Abstract: Influenza virus infection poses a significant health risk to newborns, with this population experiencing higher hospitalization and mortality rates compared to older children. The heightened vulnerability of this age group results from a combination of an altered immune system and lack of a licensed vaccine for children under six months of age. mRNA-LNP vaccines have shown remarkable efficacy, including the capacity to induce antibodies in poorly responding populations. This makes them a promising candidate for addressing the unique immunological environment of newborns. Here, we leveraged the close immunological and physiological similarity of NHP to evaluate the efficacy of an influenza hemagglutinin mRNA-LNP vaccine in newborns. Our findings show the HA mRNA-LNP vaccine elicits robust, multi-functional antibody responses in newborn NHP that result in significantly reduced viral load and disease severity following challenge. These results highlight the potential of mRNA-based vaccines as a transformative approach to protect the vulnerable newborn population against influenza. Continued development and optimization of this platform could address the critical gap in influenza virus and other pathogen vaccine coverage for infants under six months of age.

Page CL
Holbrook BC
Crofts KF
Alameh MG
Davis B
Caudell D
Weissman D
Alexander-Miller MA


mRNA vaccines targeting Leptospira immunoglobulin-like proteins confer partial protection in a hamster model of leptospirosis

Abstract: Leptospirosis is a neglected tropical disease with significant global health and economic impacts, particularly in resource-limited regions. This study reports the development of the first mRNA-based vaccines for leptospirosis, targeting the C-terminal region of LigA (LigAc) and the N-terminal region of LigB (LigBn) of Leptospira interrogans serovar Pomona. Transfection of lipid nanoparticle (LNP)-encapsulated mRNA constructs into HEK293T cells confirmed antigen expression and secretion, with proteins exhibiting higher-than-expected molecular weights due to glycosylation. In Jcl:ICR mice, LigAc- and LigBn-mRNA-LNPs elicited rapid and robust antibody responses, with significantly higher IgG titers than recombinant proteins formulated with AddaVax adjuvant. Immune sera from Lig-mRNA-LNP-vaccinated mice promoted complement-mediated killing of pathogenic leptospires in vitro. Moreover, the mRNA-LNP vaccines generated antigen-stimulated IFN-γ-ELISpot responses consistent with Th1-associated cellular activation, similar to recombinant proteins. Protective efficacy was then evaluated in golden Syrian hamsters immunized either intramuscularly or intradermally. Lig-mRNA-LNPs conferred partial protection, with a maximum survival rate of 25 % in LigBn-mRNA-LNPs vaccinated hamsters. The protective rates of Lig-mRNA-LNPs were equivalent to those of recombinant Lig protein formulations. The surviving hamsters showed reduced renal leptospiral colonization and histopathological changes, comparable to those observed in the uniformly surviving killed-leptospires group. These findings establish proof-of-concept for an mRNA vaccine platform against leptospirosis and highlight its potential application pending further optimization.

Techawiwattanaboon T
Leekitcharoenphon R
Alameh MG
Boonkea S
Sangkanjanavanich N
Nakornpakdee Y
Ajimathorn Y
Prompetchara E
Ketloy C
Buranapraditkun S
Palaga T
Kanthawong S
Heyes J
Weissman D
Ruxrungtham K
Patarakul K

Animals
Leptospirosis
Mice
Disease Models, Animal
Antibodies, Bacterial
Cricetinae
Bacterial Vaccines
Antigens, Bacterial
Humans
Vaccines, Synthetic
Immunoglobulin G
HEK293 Cells
Female
Bacterial Proteins
Leptospira
Mesocricetus
Leptospira interrogans
mRNA Vaccines
Nanoparticles
Leptospira interrogans serovar pomona
RNA, Messenger


An integrated workflow for structural virology with a 100 keV electron microscope

Abstract: Cryo-EM has revolutionized structural biology, especially for flexible and heterogeneous samples, although access to high end microscopes that enable these studies remains a bottleneck. While 300 keV microscopes have been the go-to for high-resolution structural determination, they are expensive and restricted to institutional and national facilities needing specialized expertise, with access falling far short of the demand. Here, we present the user-managed operation of a cheaper 100 keV electron microscope within a structural biology laboratory enabling close integration with protein production, biochemical and biophysical studies. We provide details and considerations for the installation of the microscope, its day-to-day maintenance, and operations. Using virus surface glycoproteins as case studies, we illustrate the workflow from grid screening, data collection, and data processing, and provide examples of data quality. This user-administered setup provides a training platform for researchers at all levels, with beginners in cryo-EM achieving proficiency to independently operate the microscope within a month of regular use and training. We have demonstrated routine high-quality low-resolution reconstructions using a Ceta CMOS camera and high-resolution reconstructions enabling building of atomic models using a Falcon C direct detector. While there are several examples of facilities that manage cryo-EM and individual laboratories leveraging cryo-EM, we provide here the first demonstration of a modern group independently doing both successfully, something that has been talked about frequently but rarely seen.

Pathirage R
Dutta M
Parsons RJ
Lella M
Atwood E
Zhang QE
May A
Johnson A
Huang X
Flemming J
Kumar U
Marayati BF
Spurrier MA
Liu C
Zhuo J
Song K
Adhikari RD
Sammour S
Ilevbare V
Abram C
Diaz M
Guzman A
Rai J
Skelly AN
Hogarty MP
Anasti K
Purro M
Lindsay M
Alam M
Weissman D
Herschchorn A
Hahn BH
Shaw GM
Sharma A
Heaton NS
Edwards RJ
Henderson R
Denny T
Saunders KO
Siliciano J
Siliciano R
Haynes BF
Janowska K
Acharya P


Macrophage cell therapy enabled by interleukin-4 mRNA-loaded lipid nanoparticles to sustain a pro-reparative phenotype in inflammatory injuries

Abstract: The use of macrophage cell therapies is limited by their tendency to change phenotype in response to external cues in situ. Here we demonstrate that an optimized lipid nanoparticle (LNP) formulation effectively delivers IL4 mRNA to human and murine primary macrophages, resulting in rapid transfection, IL-4 secretion, and reparative phenotype modulation. In a model of murine volumetric muscle loss, adoptively transferred macrophages pre-treated with IL4-LNPs maintained a reparative phenotype for at least one week, despite the inflammatory injury microenvironment. IL4-LNP-treated macrophages also promoted a reparative phenotype in endogenous macrophages and supported muscle repair outcomes, including increased vascularization, fiber size distribution, and remodeling of the scaffold. T cell subtype in the muscle or the draining lymph node was not affected. The novel strategy established here may facilitate the control and use of macrophage cell therapies for other applications in regenerative medicine.

O'Brien EM
Tylek T
Geisler HC
Mukalel AJ
Whitaker RC
Sung S
Binder-Markey BI
Weissman D
Mitchell MJ
Spiller KL

Macrophages
Animals
Interleukin-4
Nanoparticles
Humans
RNA, Messenger
Inflammation
Mice
Lipids
Phenotype
Mice, Inbred C57BL
Cell- and Tissue-Based Therapy
Liposomes


Anionic lipids modulate mRNA-lipid nanoparticle immunogenicity and confer protection in a mouse model of multiple sclerosis

Abstract: The modularity of mRNA-lipid nanoparticle (mRNA-LNP) platforms has enabled their rapid adaptation from infectious disease vaccines to emerging applications in immune-mediated disorders. However, extending mRNA-LNPs to autoimmune and inflammatory diseases requires precise control over immune cell targeting and immunogenicity. Here, we systematically investigate how incorporating anionic lipids into LNPs modulates both immune cell tropism and innate immune activation. Using a library of 40 distinct LNP formulations, we demonstrate that anionic lipids enhance mRNA delivery to splenic dendritic cells, reduce early cellular markers of adjuvant activity and tune cytokine responses in a lipid-dependent manner. We identify formulations that retain pro-inflammatory adjuvant activity and others that promote tolerogenic responses. A lead formulation containing the anionic lipid DOPG selectively dampens innate activation and induces IL-10 production. When encoding the myelin antigen MOG35-55, this LNP suppresses disease in a mouse model of multiple sclerosis, reducing neuroinflammation, T cell infiltration, and maintaining myelin morphology. These findings establish a framework for designing immune-targeted mRNA-LNPs with tunable immunogenicity and promote the development of antigen-specific tolerizing immunotherapies for autoimmune disease.

Melamed JR
Muscat-Rivera J
Kegel M
Chaboub LS
Perez-Tremble R
Bhalla NS
Ni H
Sun H
Weissman D


Consistent Induction of Broadly Neutralizing HIV Antibodies by a Novel Two-Step Mechanism Informs Immunogen Design

Abstract: A major obstacle confronting HIV-1 vaccine and cure research is the lack of an outbred animal model for rapid and consistent induction of broadly neutralizing antibodies (bNAbs). We designed an epitope-focused simian-human immunodeficiency virus (SHIV.5MUT) that elicited broad and potent V3-glycan-targeted antibodies within a year of infection in 14 of 22 macaques compared with 0 of 14 control animals. SHIV.5MUT elicited bNAbs by a novel two-step mechanism, inducing an initial wave of V1-directed antibodies that selected for Envs with shortened, hypoglycosylated V1 loops, which in turn primed V3-glycan bNAb precursors. Rhesus bNAbs were immunogenetically and structurally diverse, closely resembling human V3-glycan bNAbs. Env-bNAb coevolution revealed a diverse repertoire of bNAb precursors and the Env variants that matured them, yielding a molecular blueprint for vaccine design.

Skelly AN
Gristick HB
Li H
Gavor E
Connell AJ
Kreider EF
Marchitto L
Hogarty MP
Newby ML
Allen JD
Liu W
West AP Jr
Ayyanathan K
Campion MS
Winters K
Gordon CG
Osbaldeston RA
Akeley MJ
Li Y
Singh A
Cruickshank K
Park Y
Zhao C
Li X
Amereh K
Itallie EV
Carey JW
Albertus A
DeLaitsch AT
Keeffe JR
Lituchy MG
Walsh AA
Morris DJ
Habib R
Bibollet-Ruche F
Mishra N
Avillion G
Koranda NS
Plante SJ
Martella CL
Lora J
Wang EJD
Lewis MG
Martin MA
Nussenzweig MC
Seaman MS
Irvine DJ
Wiehe KJ
Haynes BF
Wagh K
Korber BT
Andrabi R
Crispin M
Weissman D
Bjorkman PJ
Hahn BH
Shaw GM


In vivo reprogramming of cytotoxic effector CD8 (+) T cells via fractalkine-conjugated mRNA-LNP

Abstract: Selective in vivo reprogramming of cytotoxic effector CD8 + T (T eff ) cells holds tremendous promise as a therapeutic tool but has not yet been accomplished. Here, we demonstrate that fractalkine-conjugated mRNA lipid nanoparticles (mRNA-LNP) can specifically target and deliver mRNA to CX3CR1 + T eff cells in vitro and in vivo. In mice, fractalkine-conjugated LNP target up to 90% of blood and splenic T eff cells, and delivery of IL-2-encoding mRNA to T eff cells enables robust exogenous IL-2 secretion. In rhesus macaques, fractalkine-conjugated mRNA-LNP target up to ∼100% of peripheral blood T eff cells and delivery of CD62L-mRNA enables transient CD62L expression. Collectively, these data demonstrate the potential of natural receptor ligand-based targeting of mRNA-LNP for effective and efficient transient in vivo modification of T eff cells.

Corrigan AR
Ngiow SF
Statzu M
Pampena MB
Nordin JML
Albertus A
Carro SD
Harper J
Stammen RL
Wood J
Hamilton JT
Ni H
Su J
Ramalingam R
Wu VH
Paiardini M
Weissman D
Wherry EJ
Kreider EF
Betts MR


Lipid nanoparticle co-delivery of mRNA and a small molecule drug for oral cancer chemoimmunotherapy

Abstract: Oral squamous cell carcinoma (OSCC) represents 90% of all head and neck cancers. Despite decades of research, the 5-year survival rate is 50%, and strikingly, the overall incidence rate is projected to increase by 30% in the next ten years, which will result in a sharp increase in mortality. Two fundamental aspects of OSCC are that it progresses via the inactivation and mutation of tumor suppressor (TS) genes and has a "cold" tumor immune microenvironment (TIME). A major barrier in the treatment of OSCC is the lack of novel therapies clinicians have at their disposal that are designed to disrupt tumor progression by reshaping the cold tumors into inflammatory "hot" tumors. To overcome these obstacles, we employed a lipid nanoparticle (LNP) that co-encapsulates p53 mRNA and the small molecule ciclopirox (CPX). We demonstrate that both drugs have innate chemotherapeutic properties by facilitating caspase activation. Moreover, these therapies can create a less immunosuppressive TIME in part by repolarizing tumor-associated macrophages (TAMs) to M1-like phenotypes. When formulated together, our platform provides an all-in-one approach for OSCC, effective in both p53-therapy-susceptible and p53-therapy-resistant models. Additionally, this work provides a template for a delivery platform capable of tackling multiple mechanisms of OSCC progression and survival.

Padilla MS
Li JJ
Zhang Q
Patwari K
Shi S
Yamagata HM
Joseph RA
Hymms BN
Teerdhala SV
Alameh MG
Weissman D
Le AD
Mitchell MJ


SRSF3 knockdown-induced cellular senescence as a possible therapeutic strategy for non-small cell lung cancer

Abstract: Tyrosine kinase (TK) inhibitors improve clinical outcomes in non-small cell lung cancer (NSCLC) with targetable mutations. However, such NSCLC cases account for only about 50% in the western populations. Inhibition of the splicing factor SRSF3 has been reported to be tumor-suppressive in other cancer cell types. This study for the first time explores the tumor-suppressive activity of siRNA knockdown of SRSF3 in NSCLC cells. The cell lines used were A549 (no TK mutation; TP53 wild type), NCI-H1975 (EGFR L858R/T790M; TP53 R273H mutant), NCI-H322 (no TK mutation; TP53 R248L mutant), and NCI-H596 (no TK mutation; TP53 G245C mutant). In all these cell lines, SRSF3 knockdown increased cellular senescence, as indicated by increased senescence-associated β-galactosidase activity and reduced cell proliferation. In A549 cells, increased apoptotic cleavage of caspase-3 and poly(ADP-ribose) polymerase was also observed. A tumor-suppressive p53 isoform, p53β, was shown to be upregulated by SRSF3 knockdown. However, overexpression of p53β did not induce cellular senescence or apoptosis, suggesting that this p53 isoform is not a primary effector of SRSF3 knockdown in NSCLC cells. Gene expression analyses suggested that the SRSF3 knockdown-induced senescence in NSCLC cells may be mediated by the downregulation of TOP2A, UBE2C, or ASPM, which are known oncogenic factors associated with poor patient prognosis. We also generated SRSF3 siRNA-encapsulating lipid nanoparticles as a future therapeutic tool. This study proposes a therapeutic strategy for NSCLC that is independent of the mutation status of TP53 and TK-encoding genes.

Nakamichi S
von Muhlinen N
Yamada L
Melamed JR
Papp TE
Parhiz H
Weissman D
Horikawa I
Harris CC

Humans
Carcinoma, Non-Small-Cell Lung
Serine-Arginine Splicing Factors
Cellular Senescence
Lung Neoplasms
Cell Proliferation
Apoptosis
Tumor Suppressor Protein p53
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
RNA, Small Interfering
A549 Cells
Gene Knockdown Techniques
Mutation

Journal Article
Research Support, N.I.H., Intramural


Hypercontractility and Oxidative Stress Drive Creatine Kinase Dysfunction in Hypertrophic Cardiomyopathy

Abstract: Hypertrophic cardiomyopathy (HCM) is a prevalent inherited cardiac disorder marked by left ventricular hypertrophy and hypercontractility. This excessive mechanical workload creates an energetic mismatch in which consumption exceeds production, leading to myocardial energy depletion. Although CK (creatine kinase) plays a key role in cardiac energy homeostasis, its involvement in HCM remains unclear. This study investigates how hypercontractility-driven mitochondrial stress and the resulting increase in mitochondrial H2O2 disrupt CK function in HCM. CK function was analyzed using myocardial left ventricular tissue from 92 patients with HCM (with and without pathogenic sarcomere variants) and 30 non-failing human controls. Myofilament and mitochondrial CK isoforms were measured using mRNA analysis, protein immunoblotting, enzyme activity assays, mass spectrometry, and redox-sensitive proteomics. To explore links between hypercontractility, mitochondrial reactive oxygen species, and CK dysfunction, we used isolated cardiomyocytes from wild-type, mitochondrial-targeted catalase-overexpressing, CK knockout (myofilament and mitochondrial CK deletion), HCM-associated Mybpc3 knock-in, and mito-roGFP2-Orp1 mouse models. We also tested the effects of the Ca2+ sensitizer EMD-57033, the CK inhibitor 1-fluoro-2,4-dinitrobenzene (DNFB), and the myosin inhibitor MYK-581, a mavacamten derivative. Our analysis revealed significant reductions in myofilament and mitochondrial CK protein levels, as well as CK activity, in myocardium of patients with HCM, primarily because of oxidative modifications of CK. In isolated mouse cardiomyocytes from wild-type and CK knockouts, hypercontractility induced by EMD-57033 elevated mitochondrial H2O2, causing cellular arrhythmias and CK inactivation. Hypercontractility-induced oxidative stress, arrhythmias, and CK dysfunction were also observed in Mybpc3 knock-in cardiomyocytes. Mitochondrial-targeted catalase-overexpressing mice with enhanced H2O2 scavenging were protected against H2O2-induced (EMD-57033-mediated) arrhythmias and CK dysfunction. MYK-581 treatment in Mybpc3 knock-in cardiomyocytes reduced hypercontractility, lowered H2O2 production and arrhythmias, and preserved CK function. CK inhibition using DNFB in wild-type cardiomyocytes elevated mitochondrial H2O2 levels and triggered cellular arrhythmias. This mitochondrial oxidation was independently confirmed in mito-roGFP2-Orp1 cardiomyocytes exposed to DNFB. Mitochondrial-targeted catalase-overexpressing mice were protected from DNFB-induced oxidative stress and arrhythmogenic events. This study reveals a mechanistic link between hypercontractility, mitochondrial reactive oxygen species, and CK dysfunction in HCM, perpetuating a cycle of energetic dysfunction. Targeting hypercontractility and oxidative stress through myosin inhibition offers a strategy to restore energy balance and reduce arrhythmic risk in HCM.

Xu A
Weissman D
Ermer KJ
Bertero E
Federspiel JM
Stadler F
Grünler E
Tangos M
Zervou S
Waddingham MT
Pearson JT
Reil JC
Scholtz S
Dudek J
Kohlhaas M
Nickel AG
Carrier L
Eschenhagen T
Michels M
Dos Remedios C
Lal S
Prates Roma L
Hamdani N
Kuster DWD
Falcão-Pires I
Johnson CN
Lygate CA
van der Velden J
Maack C
Sequeira V

Animals
Oxidative Stress
Humans
Cardiomyopathy, Hypertrophic
Male
Myocytes, Cardiac
Mice
Female
Middle Aged
Creatine Kinase
Mitochondria, Heart
Myocardial Contraction
Adult
Mice, Knockout
Mice, Inbred C57BL
Hydrogen Peroxide
Carrier Proteins
Myosin Binding Protein C

pmc-id: PMC13227915;manuscript-id: NIHMS2115955;embargo-date: 2026/10/20;


In vivo chimeric antigen receptor (CAR)-T cell therapy

Abstract: Chimeric antigen receptor (CAR)-T cell therapy has transformed the outcomes of patients with haematological malignancies, yet its use is limited by labour-intensive manufacturing, constrained production capacity and variable clinical performance. In vivo CAR-T cell engineering, in which CAR-T cells are generated directly inside the patient's body, seeks to overcome these challenges by eliminating the need for ex vivo cell processing and complex logistics, as well as improve clinical performance. Recent advances in virology, RNA medicines and nanotechnology have catalysed a radical overhaul of this approach, which uses targeted delivery systems such as lentiviral vectors and lipid nanoparticles to introduce CAR-encoding genetic material into endogenous T cells. Early clinical studies have shown efficient transduction, sustained CAR expression and initial signs of antitumour activity, establishing proof of concept. This Review explores the underlying technologies - including RNA delivered by lipid nanoparticles and engineered viral vectors - and discusses how they are being adapted to develop more broadly applicable, scalable, safe and effective CAR-T cell therapies. By removing the need for ex vivo manipulation and chemotherapeutic conditioning, this strategy could enable the wider application of CAR-T cell therapies not just to blood cancers but to autoimmune diseases for which ex vivo CAR-T cell therapies have shown strong promise, such as systemic lupus erythematosus.

Bot A
Scharenberg A
Friedman K
Guey L
Hofmeister R
Andorko JI
Klichinsky M
Neumann F
Shah JV
Swayer AJ
Trudeau K
Weissman D
Stephan MT
Buchholz CJ
June CH

Humans
Receptors, Chimeric Antigen
Animals
Immunotherapy, Adoptive
T-Lymphocytes
Nanoparticles
Genetic Vectors
Hematologic Neoplasms


Allergen-specific mRNA-lipid nanoparticle therapy for prevention and treatment of experimental allergy in mice

Abstract: Allergic diseases have reached epidemic proportions globally, calling attention to the need for better treatment and preventive approaches. Herein, we developed allergen-encoding messenger RNA (mRNA)-lipid nanoparticle (LNP) strategies for both therapy and prevention of allergic responses. Immunization with allergen-encoded mRNA-LNPs modulated T cell differentiation, inhibiting the generation of T helper type 2 and type 17 cells upon allergen exposure in experimental asthma models induced by ovalbumin, and naturally occurring house dust mite (HDM) and the major HDM allergen Der p1. Allergen-specific mRNA-LNP treatment attenuated clinicopathology in both preventive and established allergy models, including reduction in eosinophilia, mucus production, and airway hypersensitivity, while enhancing production of allergen-specific IgG antibodies and maintaining low IgE levels. Additionally, allergen-specific mRNA-LNP vaccines in mice elicited a CD8+CD38+KLRG- T cell response as seen following SARS-CoV-2 mRNA vaccination in humans, underscoring a conserved immune mechanism across species, regardless of the mRNA-encoded protein. Notably, mRNA-LNP vaccination in combination with an mTOR inhibitor reduced the CD8+ T cell response without affecting the vaccine-induced anti-allergic effect in the preventive model of asthma. This technology renders allergen-specific mRNA-LNP therapy a promising approach for prevention and treatment of allergic diseases.

Rochman Y
Kotliar M
Klingler AM
Rochman M
Alameh MG
Melamed JR
Osswald GA
Caldwell JM
Felton JM
Mack LE
Hargis J
Lewkowich IP
Barski A
Weissman D
Rothenberg ME

Animals
Mice
Nanoparticles
Asthma
RNA, Messenger
Allergens
Antigens, Dermatophagoides
Disease Models, Animal
Female
Humans
Lipids
Arthropod Proteins
Mice, Inbred BALB C
Pyroglyphidae
SARS-CoV-2
Ovalbumin
COVID-19 Vaccines
Hypersensitivity
Liposomes

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't


Harnessing mRNA-lipid nanoparticles as innovative therapies for autoimmune diseases

Abstract: Autoimmune diseases are chronic conditions where the immune system mistakenly attacks healthy tissues, leading to potentially debilitating symptoms that require lifelong management. There are no cures for autoimmune diseases, and new treatments are urgently needed to improve patient outcomes. This review delves into the compelling advancements and ongoing challenges in harnessing mRNA-lipid nanoparticles (LNPs) as innovative therapies for autoimmune diseases. mRNA-LNPs enable a range of therapeutic approaches to combat autoimmune diseases, including targeted immune cell modulation, tissue regeneration, antigen-specific tolerizing immunotherapy, and in vivo chimeric antigen T cell therapies. To successfully advance this promising class of therapies to the clinic, key challenges must be addressed, such as mitigating unwanted inflammation caused by LNPs, overcoming biological barriers to delivery, and ensuring the long-term safety of mRNA-LNPs specifically in autoimmune contexts. Through their modular design, flexible application, and potential for cost-effective production, mRNA-LNP therapies offer exciting clinical potential to transform the management of autoimmune diseases.

Molecular therapy. Methods & clinical development

Razavi R
Kegel M
Muscat-Rivera J
Weissman D
Melamed JR


Hepatitis C virus modified (S)E2(F442NYT)-mRNA-LNP candidate vaccine promotes helper CXCR5(+)T cells

Abstract: T cells play an important role in initiating antibody responses by instructive signals of cell-cell contacts and secretion of soluble cytokines as mediators. We investigated the role of the modified soluble E2 (sE2F442NYT) antigen from hepatitis C virus (HCV) on healthy human peripheral blood mononuclear cell (PBMC)-derived immune cells or immunized mouse cells to understand the mechanisms of immune regulation by the candidate vaccine antigen. HCV E2 and E2F442NYT displayed a role in inducing type 17 T-helper cell (Th17) phenotype, as indicated by interleukin-17 (IL-17) expression and signal transducer and activator of transcription 3 (Stat3) phosphorylation. The spleen cells from sE2-mRNA-lipid nanoparticles (LNPs) or sE2F442NYT-mRNA-LNP-immunized mice exhibited similar IL-17A mRNA levels, and Th17 (CXCR3-CCR6+) cells in CD4+CD44+ spleen cells, supporting both sE2 and modified sE2F442NYT-induced Th17 polarization. Immunohistochemical and multiplex immunofluorescence imaging studies revealed abundant CD4+CXCR5+T cells co-localized with BCL6 in sE2F442NYT-mRNA-LNP immunized mouse spleen cells than unmodified sE2-mRNA-LNP immunized animals, suggesting sE2F442NYT induces stronger follicular helper T cell generation. We previously demonstrated increased total IgG production and isotype switching from IgG1 to IgG2a and IgG2b in sE2442NYT immunized mice. The stronger B and T cell responses observed from modified sE2F442NYT support the overall in vivo outcome of the study toward a higher B helper T cell generation from sE2F442NYT-mRNA-LNP immunization as compared to unmodified sE2-mRNA-LNP.IMPORTANCEThe study will help rationalize HCV vaccine antigen selection for an effective immune response. Extension by additional strategies may be useful to direct stronger B helper T cell generation for prolonged vaccine-associated protection.

Haga Y
Babu E P
Swiderska-Syn M
Reagan EK
Weissman D
Ray R

Animals
Humans
Mice
Hepacivirus
Th17 Cells
Receptors, CXCR5
Viral Hepatitis Vaccines
RNA, Messenger
Nanoparticles
Female
Interleukin-17
Hepatitis C
Leukocytes, Mononuclear
T-Lymphocytes, Helper-Inducer
Liposomes
Viral Envelope Proteins

Journal Article
Research Support, N.I.H., Extramural


Immunogenicity and safety of 'Comvigen', a bivalent SARS-CoV-2 vaccine, in comparison to Comirnaty bivalent vaccine in Thailand: a phase 2, non-inferiority randomised trial

Abstract: Strengthening mRNA vaccine development in LMICs is essential for enhancing global pandemic preparedness. This study evaluated the safety and immunogenicity of Comvigen, a bivalent SARS-CoV-2 vaccine, in comparison to the Comirnaty bivalent vaccine (Comirnaty). This phase II, randomised, open-label, non-inferiority trial was conducted in Thailand across four centres. Participants (n = 450) were randomly assigned (2:1) to receive either Comvigen (50 μg) or Comirnaty (30 μg), using block randomisation (size = 9). Eligible participants had completed at least 2 doses of any approved COVID-19 vaccine, with the last mRNA-vaccine dose given over 3 months before enrolment. The non-inferiority margin of a geometric mean ratio (GMR) of 0.67. The primary immunogenicity endpoint was pseudovirus neutralisation titres (psVNT-50) against SARS-CoV-2 wild-type and Omicron BA.4/BA.5 at Day 29. Safety outcomes included local and systemic adverse reactions up to six months post-vaccination. Immunogenicity analyses were conducted on the Per-Protocol (PP) population and the modified Intent-to-Treat (mITT) population; safety analyses included all participants. Laboratory personnel were blinded to vaccine assignment (ClinicalTrials.gov: NCT05930730). Between October and November 2023, 450 participants were enrolled (median age of 36 years, IQR 30-45). At day 29, the geometric mean titre (GMT) of psVNT-50 against wild-type virus increased from 475.9 to 2062.9 for Comvigen and from 458.8 to 1905.1 for Comirnaty (GMR 1.1, 95% CI: 1.0-1.2), meeting non-inferiority criteria. Against Omicron BA.4/BA.5, GMTs were 3909.8 for Comvigen and 3288.6 for Comirnaty (GMR 1.2, 95% 1.0-1.4). Local and systemic reactions were more frequent with Comvigen (91% vs. 78%, p = 0.0002, 79% vs. 70%, p = 0.028) but were mild or moderate and transient with no difference in fever (6% vs. 5%, p = 0.84). Comvigen demonstrated non-inferiority immunogenicity to Comirnaty and had a comparable safety profile, supporting mRNA vaccine development for global access and pandemic preparedness. Covid-19 Pandemic Emergency Fund granted by Thailand's National Economic and Social Development Council provided major funding. Supplementary funding was provided by National Vaccine Institute (NVI), Thailand; Center of Excellence in Vaccine Research and Development (Chula VRC), Faculty of Medicine, Chulalongkorn University; Chulalongkorn University Second Century Fund (C2F); BioNet-Asia and Public Donation through Covid-19 vaccine development fund of the Faculty of Medicine, Chulalongkorn University and the Thai Red Cross Society, Thailand.

The Lancet regional health. Southeast Asia

Jantarabenjakul W
Nantanee R
Puthanakit T
Gatechompol S
Avihingsanon A
Punrin S
Tantawichien T
Nitayaphan S
Thitithanyanont A
Buranapraditkun S
Jongkaewwattana A
Ketloy C
Prompetchara E
Lawpoolsri S
Wijagkanalan W
Alameh MG
Hong L
Samija M
Weissman D
Ruxrungtham K
ChulaVac006 Study Team


Homologous and Heterologous Vaccination Regimens with mRNA and rVSV Platforms Induce Potent Immune Responses Against SFTSV Glycoprotein

Abstract: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic bunyavirus with a high case-fatality ratio for which there is no approved vaccine. Studies have assessed different vaccine technologies. However, few studies have yet assessed the immunogenicity of heterologous prime-boost regimens. Here, we compare a lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA-based vaccine encoding the SFTSV glycoproteins, Gn and Gc, to our recently described recombinant VSV SFTSV (rVSV-SFTSV) vaccine in single dose, homologous, and heterologous prime-boost regimens in mice. We show that all regimens protect from pathogenic SFTSV challenge and elicit strong long-lasting antibody responses. Furthermore, strong cellular immunity is elicited by mRNA-LNP immunizations and by heterologous immunization with an rVSV-SFTSV prime and mRNA-LNP boost. Cellular responses robustly polarized towards a type 1 response, characterized by high levels of IFNγ, TNFα, and IL-2. Immunization with mRNA led to a mixed type 1/type 2 immune response, as determined by antibody isotypes IgG1 and IgG2c. We found that homologous immunization leads to stronger antibody responses while heterologous immunization drives a slightly stronger cellular response. Taken together, the vaccine platforms described here represent strong vaccine candidates for further development.

Manzoni TB
Westover JB
Lundgreen KA
Hicks PD
Petch RJ
Ort JT
Weissman D
Fan SHY
Hensley SE
Pardi N
Gowen BB
Bates P

Severe Fever with Thrombocytopenia Syndrome


mRNA-LNP vaccine encoding the Plasmodium vivax circumsporozoite protein is highly immunogenic and confers protection in mice

Abstract: Plasmodium vivax poses significant challenges to malaria control due to its relapsing nature. This study explores the immunogenicity and efficacy of nucleoside-modified mRNA-lipid nanoparticle (LNP) vaccines targeting the P. vivax circumsporozoite protein (PvCSP). Two mRNA constructs encoding PvCSP were designed and tested in mice. Despite lower protein expression, the vaccine encoding the wild-type signal peptide (SP) and glycosylphosphatidylinositol (GPI) anchor of PvCSP induced significantly higher antibody titers against the PvCSP and its repeat region compared with the mRNA construct with SP but without GPI. The immunogenicity of PvCSP mRNA-LNP vaccines was evaluated using various administration routes and immunization schedules. Both intradermal and intramuscular delivery generated dose-dependent antibody responses, but the former demonstrated superior responses at a lower dose. Conversely, intravenous administration resulted in very poor responses. Notably, administering a delayed third dose intramuscularly 5 months after the second dose resulted in significantly higher levels of anti-repeat region antibodies and enhanced T cell responses in both the spleen and liver. This delayed regimen provided strong protection against sporozoite challenge, with the magnitude and avidity of anti-repeat region antibodies linked to this protection. These findings highlight the potential of the nucleoside-modified mRNA-LNP vaccine platform in combating P. vivax pre-erythrocytic stage infection.

Limsalakpetch A
Kum-Arb U
Yongvanitchit K
Im-Erbsin R
Ubalee R
Waters N
Vesely BA
Muramatsu H
Weissman D
Tam YK
Yoshida S
Adams J
Yadava A
Pardi N
Pichyangkul S


Author Correction: Mannich reaction-based combinatorial libraries identify antioxidant ionizable lipids for mRNA delivery with reduced immunogenicity

Gong N
Kim D
Alameh MG
El-Mayta R
Han EL
Dwivedi G
Palanki R
Shi Q
Han X
Xue L
Xu J
Meng Z
Luo T
Figueroa-Espada CG
Weissman D
Li J
Mitchell MJ


ATP-Citrate Lyase in Heart Failure: A Redox Node in NAD(+)/NADH Homeostasis


Mannich reaction-based combinatorial libraries identify antioxidant ionizable lipids for mRNA delivery with reduced immunogenicity

Abstract: The immunogenicity of lipid nanoparticles (LNPs) used for the delivery of nucleoside-modified messenger RNA limits the levels and durability of expression of the encoded protein. Here, by leveraging the Mannich reaction for ionizable lipid synthesis, and via the in vitro and in vivo screening of six combinatorial libraries of synthesized lipids, we report the identification of an antioxidant ionizable lipid, C-a16, exhibiting reduced immunogenicity. When incorporated into LNPs for mRNA delivery, C-a16 mitigated the generation of intracellular reactive oxygen species, thereby extending the duration of protein expression. In mice, and compared with commercial LNPs, LNPs incorporating C-a16 and co-delivering Cas9 mRNA and guide RNA for the editing of the transthyretin gene led to 2.8-fold higher editing efficiency; LNPs with C-a16 delivering fibroblast growth factor 21 mRNA increased the expression of the protein 3.6-fold; and when delivering mRNA encoding a tumour neoantigen or the spike protein of SARS-CoV-2, LNPs with C-a16 induced stronger antigen-specific immune responses. Our findings support the further testing of C-a16 as a promising ionizable lipid for mRNA delivery in therapeutic applications.

Animals
Lipids
Mice
RNA, Messenger
Humans
Nanoparticles
Antioxidants
SARS-CoV-2
Spike Glycoprotein, Coronavirus
COVID-19
Female
Reactive Oxygen Species
Gene Transfer Techniques
Combinatorial Chemistry Techniques
Gene Editing
Liposomes


Targeting DNA-LNPs to Endothelial Cells Improves Expression Magnitude, Duration, and Specificity

Abstract: DNA-lipid nanoparticles (DNA-LNPs) loaded with inhibitors of the cGAS-STING pathway enable safe and effective delivery of DNA in vivo . Herein, we report the first instances of extrahepatic DNA-LNP targeting. DNA-LNPs conjugated to antibodies against PECAM-1 or VCAM-1 target the endothelium of the lungs and brain/spleen, respectively. These LNPs drive robust transgene expression in their target organs, with greater magnitude and duration than untargeted LNPs. Lung specificity of PECAM-targeted transgene expression increases over two weeks, resulting in markedly higher lung-to-liver expression ratios than our previous PECAM-targeted mRNA-LNPs. Off-target liver DNA expression declines to undetectable levels but persists in the lungs, while mRNA expression uniformly decreases due to its short half-life. We further improve this expression specificity by replacing full-length antibodies with Fab fragments. Single-cell analysis reveals a key mechanism underlying the improvements in organ-specificity: target organ expression is dominated by long-lived endothelial cells, while off-target liver delivery and expression are in non-endothelial cells with shorter half-lives. Collectively, these studies demonstrate that targeted DNA-LNPs achieve high levels of organ- and cell-type-specific transgene expression and thus provide a therapeutic platform for dozens of endothelial-centric diseases.

Marzolini N
Brysgel TV
Rahman RJ
Essien EO
Nwe SY
Wu J
Majumder A
Patel MN
Tiwari S
Espy CL
Dong F
Shah A
Shuvaev VV
Hood ED
Chase LS
Weissman D
Katzen JB
Frank DB
Bennett ML
Marcos-Contreras OA
Myerson JW
Muzykantov VR
Reyes-Esteves S
Brenner JS


IL-12 mRNA-LNP promotes dermal resident memory CD4(+) T cell development

Abstract: Dermal resident memory CD4+ T cells (dTrm) provide protection against vector-borne infections. However, the factors that promote their development remain unclear. We tested if an mRNA vaccine, encoding a protective leishmanial antigen, induced dTrm cells. The mRNA vaccine induced robust systemic T-cell responses, but few Trm cells were found in the skin. Since IL-12 promotes Th1 responses, we tested whether IL-12 mRNA combined with the mRNA vaccine could enhance dTrm cell development. This combination significantly expanded Leishmania-specific Th1 cells expressing skin-homing molecules and memory T cell markers in the draining lymph node. Additionally, higher numbers of dTrm cells were maintained in the skin, and mice exhibited functional immunity indicated by a delayed hypersensitivity response and protection upon challenge with Leishmania. These findings highlight IL-12 as a key driver of CD4+ dTrm development, enabling their global seeding across the skin, and underscore the potential of IL-12-enhanced mRNA vaccines to generate durable immunity against cutaneous leishmaniasis and other skin-targeted infections.

Zabala-Peñafiel A
Gonzalez-Lombana C
Alameh MG
Sacramento LA
Mou Z
Phan AT
Aunins EA
Tam YK
Uzonna JE
Weissman D
Hunter CA
Scott P


A cargo receptor entrapment complex is a therapeutic node for genetically and clinically distinct proteinopathies

Abstract: Severe proteinopathies-such as retinitis pigmentosa, a form of inherited blindness-are driven by genetic mutations that overwhelm the quality control of the post-endoplasmic reticulum (post-ER) secretory pathway, causing toxic protein accumulation. Here, we identify a therapeutic node defined by a hetero-oligomeric cargo receptor complex consisting of TMED7, 2, 9, and 10. This "entrapment complex" anchors structurally and functionally diverse mutant clients within the early secretory pathway via TMED7 binding to the integral Golgi protein GRASP55. Disruption of the entrapment complex results in the clearance of accumulated protein cargoes. In vivo ablation of the entrapment node via inducible genetic deletion or via the small molecule BRD7635 reverses histopathological hallmarks and rescues functional deficits in clinically distinct proteinopathies of the kidney and the eye, including mitigating vision loss in a mouse model of retinitis pigmentosa.

Khursigara MR
Goss AC
Kost-Alimova M
Keller K
De Mata CD
Muraleedharan R
Collantes ER
Brown M
Grinkevich E
Arines FM
Lin J
Byrne P
Valenti SB
Morici E
Roignot J
Zavras J
Silverman BR
Ignacio JC
Myung Y
Kwon S
Nelson A
Yoo H
Melanson M
Racette M
Padovano V
Alper SL
Carey D
Udeshi ND
Carr SA
Dvela-Levitt M
Narimatsu T
Sakuno G
Correa VSMC
Efstathiou NE
Ntentakis DP
Cao T
Dong Z
Nguyen KT
Menezes CR
Kurumbail R
Kazmirski S
Xiao L
Wu H
Liu D
Iqbal S
Lander ES
Vavvas DG
Palczewski K
Pablo JLB
Greka A


Ancient DNA reveals pervasive directional selection across West Eurasia

Abstract: Ancient DNA has transformed our understanding of population history1, but its potential to reveal as much about human evolutionary biology has not been realized because of limited sample sizes and the difficulty of distinguishing sustained rises in allele frequency increasing fitness-directional selection-from shifts due to migrations, population structure, or non-adaptive purifying or stabilizing selection2-7. Here we present a method for detecting directional selection in ancient DNA time-series data that tests for consistent trends in allele frequency change over time, and apply it to 15,836 West Eurasians (10,016 with new data). Previous work has shown that classic hard sweeps driving advantageous mutations to fixation have been rare over the broad span of human evolution8,9. By contrast, in the past ten millennia, we find that many hundreds of alleles have been affected by strong directional selection. We also document one-standard-deviation changes on the scale of modern variation in combinations of alleles that today predict complex traits. This includes decreases in predicted body fat and schizophrenia, and increases in measures of cognitive performance. These effects were measured in industrialized societies, and it remains unclear how these relate to phenotypes that were adaptive in the past. We estimate selection coefficients at 9.7 million variants, enabling study of how Darwinian forces couple to allelic effects and shape the genetic architecture of complex traits.

Akbari A
Perry A
Barton AR
Kariminejad M
Gazal S
Li Z
Zeng Y
Mittnik A
Patterson N
Mah M
Zhou X
Price AL
Lander ES
Pinhasi R
Rohland N
Mallick S
Reich D

Humans
Alleles
Asia
DNA, Ancient
Europe
Evolution, Molecular
Gene Frequency
History, Ancient
Phenotype
Selection, Genetic


Functional dissection of complex trait variants at single-nucleotide resolution

Abstract: Identifying the causal variants and mechanisms that drive complex traits and diseases remains a core problem in human genetics1-5. Most of these variants individually have weak effects6 and lie in non-coding gene-regulatory elements7-10, for which we lack a complete understanding of how single-nucleotide alterations modulate transcriptional processes to affect human phenotypes5,11-15. To address this problem, we measured the activity of 221,412 fine-mapped trait-associated variants using a massively parallel reporter assay16-20 in 5 diverse cell types. We show that this assay effectively discriminates between likely causal variants and controls, and identified 13,121 regulatory variants with high precision. Although the effects of these variants largely agree with orthogonal measures of function, only 69% of them can plausibly be explained by the disruption of a known transcription factor binding motif. We investigated the mechanisms of 136 variants using saturation mutagenesis and assigned affected transcription factors for 91% of variants without a clear canonical mechanism. Finally, we detected regulatory epistasis at 11% of tested regulatory variants in close proximity and identified multiple functional variants on the same haplotype at a small, but important, subset of trait-associated loci. Overall, our study provides a systematic functional characterization of likely causal common variants that underlie complex and molecular human traits, enabling new insights into the regulatory grammar underlying disease risk.

Siraj L
Castro RI
Dewey HB
Kales S
Butts JC
Nguyen TTL
Kanai M
Berenzy D
Mouri K
Wang QS
Fiziev PP
Tsuo K
McCaw ZR
Gosai SJ
Aguet F
Cui R
Kassam I
McRae J
Vockley CM
Lareau CA
Abramov S
Boystov A
Vierstra J
Okada Y
Gusev A
Jones TR
Lander ES
Sabeti PC
Finucane HK
Reilly SK
Ulirsch JC
Tewhey R


Efficiently quantifying dependence in massive scientific datasets using InterDependence Scores

Abstract: Large-scale scientific datasets today contain tens of thousands of random variables across millions of samples (for example, the RNA expression levels of 20,000 protein-coding genes across 30 million single cells). Being able to quantify dependencies between these variables would help us discover novel relationships between variables of interest. Simple measures of dependence, such as Pearson correlation, are fast to compute, but limited in that they are designed to detect linear relationships between variables. Complex measures are known with the ability to detect any kind of dependence, but they do not readily scale to many modern datasets of interest. We introduce the InterDependence Score (IDS), a scalable measure of dependence that captures linear and various nonlinear dependencies between random variables. Our IDS algorithm is motivated by a dependence measure defined in infinite-dimensional Hilbert spaces, capable of capturing any type of dependence, and a fast (linear time) algorithm that neural networks natively implement to compute dependencies between random variables. We apply IDS to identify 1) relevant variables for predictive modeling tasks, 2) sets of words forming topics from millions of documents, and 3) sets of genes related to "gene-expression programs" in tens of millions of cells. We provide an efficient implementation that computes IDS between billions of pairs of variables across millions of samples in several hours on a single GPU. Given its speed and effectiveness in identifying nonlinear dependencies, we envision IDS will be a valuable tool for uncovering insights from scientific data.

Radhakrishnan A
Jain Y
Uhler C
Lander ES


Rewriting regulatory DNA to dissect and reprogram gene expression

Abstract: Regulatory DNA provides a platform for transcription factor binding to encode cell-type-specific patterns of gene expression. However, the effects and programmability of regulatory DNA sequences remain difficult to map or predict. Here, we develop variant effects from flow-sorting experiments with CRISPR targeting screens (Variant-EFFECTS) to introduce hundreds of designed edits to endogenous regulatory DNA and quantify their effects on gene expression. We systematically dissect and reprogram 3 regulatory elements for 2 genes in 2 cell types. These data reveal endogenous binding sites with effects specific to genomic context, transcription factor motifs with cell-type-specific activities, and limitations of computational models for predicting the effect sizes of variants. We identify small edits that can tune gene expression over a large dynamic range, suggesting new possibilities for prime-editing-based therapeutics targeting regulatory DNA. Variant-EFFECTS provides a generalizable tool to dissect regulatory DNA and to identify genome editing reagents that tune gene expression in an endogenous context.

Martyn GE
Montgomery MT
Jones H
Guo K
Doughty BR
Linder J
Bisht D
Xia F
Cai XS
Chen Z
Cochran K
Lawrence KA
Munson G
Pampari A
Fulco CP
Sahni N
Kelley DR
Lander ES
Kundaje A
Engreitz JM

Clustered Regularly Interspaced Short Palindromic Repeats


Transcription factor networks disproportionately enrich for heritability of blood cell phenotypes

Abstract: Most phenotype-associated genetic variants map to noncoding regulatory regions of the human genome, but their mechanisms remain elusive in most cases. We developed a highly efficient strategy, Perturb-multiome, to simultaneously profile chromatin accessibility and gene expression in single cells with CRISPR-mediated perturbation of master transcription factors (TFs). We examined the connection between TFs, accessible regions, and gene expression across the genome throughout hematopoietic differentiation. We discovered that variants within TF-sensitive accessible chromatin regions in erythroid differentiation, although representing <0.3% of the genome, show a ~100-fold enrichment for blood cell phenotype heritability, which is substantially higher than that for other accessible chromatin regions. Our approach facilitates large-scale mechanistic understanding of phenotype-associated genetic variants by connecting key cis-regulatory elements and their target genes within gene regulatory networks.

Martin-Rufino JD
Caulier A
Lee S
Castano N
King E
Joubran S
Jones M
Goldman SR
Arora UP
Wahlster L
Lander ES
Sankaran VG

Humans
Transcription Factors
Gene Regulatory Networks
Chromatin
Phenotype
Single-Cell Analysis
CRISPR-Cas Systems
Genome, Human
Genetic Variation
Blood Cells
Cell Differentiation
Erythropoiesis
Erythroid Cells


A standing platform for cancer drug development using ctDNA-based evidence of recurrence

Abstract: The time required to conduct clinical trials limits the rate at which we can evaluate and deliver new treatment options to patients with cancer. New approaches to increase trial efficiency while maintaining rigor would benefit patients, especially in oncology, in which adjuvant trials hold promise for intercepting metastatic disease, but typically require large numbers of patients and many years to complete. We envision a standing platform - an infrastructure to support ongoing identification and trial enrolment of patients with cancer with early molecular evidence of disease (MED) after curative-intent therapy for early-stage cancer, based on the presence of circulating tumour DNA. MED strongly predicts subsequent recurrence, with the vast majority of patients showing radiographic evidence of disease within 18 months. Such a platform would allow efficient testing of many treatments, from small exploratory studies to larger pivotal trials. Trials enrolling patients with MED but without radiographic evidence of disease have the potential to advance drug evaluation because they can be smaller (given high probability of recurrence) and faster (given short time to recurrence) than conventional adjuvant trials. Circulating tumour DNA may also provide a valuable early biomarker of treatment effect, which would allow small signal-finding trials. In this Perspective, we discuss how such a platform could be established.

Medford AJ
Carmeli AB
Ritchie A
Wagle N
Garraway L
Lander ES
Parikh A

Humans
Circulating Tumor DNA
Neoplasms
Neoplasm Recurrence, Local
Biomarkers, Tumor
Drug Development
Antineoplastic Agents
Clinical Trials as Topic


Pervasive findings of directional selection realize the promise of ancient DNA to elucidate human adaptation

Abstract: We present a method for detecting evidence of natural selection in ancient DNA time-series data that leverages an opportunity not utilized in previous scans: testing for a consistent trend in allele frequency change over time. By applying this to 8433 West Eurasians who lived over the past 14000 years and 6510 contemporary people, we find an order of magnitude more genome-wide significant signals than previous studies: 347 independent loci with >99% probability of selection. Previous work showed that classic hard sweeps driving advantageous mutations to fixation have been rare over the broad span of human evolution, but in the last ten millennia, many hundreds of alleles have been affected by strong directional selection. Discoveries include an increase from ~0% to ~20% in 4000 years for the major risk factor for celiac disease at HLA-DQB1; a rise from ~0% to ~8% in 6000 years of blood type B; and fluctuating selection at the TYK2 tuberculosis risk allele rising from ~2% to ~9% from ~5500 to ~3000 years ago before dropping to ~3%. We identify instances of coordinated selection on alleles affecting the same trait, with the polygenic score today predictive of body fat percentage decreasing by around a standard deviation over ten millennia, consistent with the "Thrifty Gene" hypothesis that a genetic predisposition to store energy during food scarcity became disadvantageous after farming. We also identify selection for combinations of alleles that are today associated with lighter skin color, lower risk for schizophrenia and bipolar disease, slower health decline, and increased measures related to cognitive performance (scores on intelligence tests, household income, and years of schooling). These traits are measured in modern industrialized societies, so what phenotypes were adaptive in the past is unclear. We estimate selection coefficients at 9.9 million variants, enabling study of how Darwinian forces couple to allelic effects and shape the genetic architecture of complex traits.

Akbari A
Barton AR
Gazal S
Li Z
Kariminejad M
Perry A
Zeng Y
Mittnik A
Patterson N
Mah M
Zhou X
Price AL
Lander ES
Pinhasi R
Rohland N
Mallick S
Reich D