Reviewing clinical considerations and guideline recommendations of C1 inhibitor prophylaxis for hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE), a rare disease that is characterized by painful and recurring non-allergic swelling episodes, is caused by the deficiency or dysfunction of C1 inhibitor (C1INH) protein. A comprehensive HAE management plan may require long-term prophylaxis (LTP) in addition to on-demand treatment to help "normalize" patients' lives so that they may fully engage in work, school, family, and leisure activities. AIM: The main objective of this narrative review is to provide an overview of updated guideline recommendations specific to LTP of HAE and discuss clinical considerations and pharmacologic management options, with a focus on C1INH. MATERIALS AND METHODS: The authors reviewed relevant HAE literature for current recommendations regarding LTP and the role of C1NH. RESULTS: Acute HAE attacks are treated with on-demand medication; however, there is a consensus that LTP should routinely be considered for risk reduction and prevention of future episodes. The 2017 World Allergy Organization/European Academy of Allergy and Clinical Immunology guidelines recommend that all patients with HAE be evaluated for LTP routinely and the 2020 HAE Association (HAEA) guidelines emphasize that the decision to use LTP should not be based on rigid criteria, but rather should be based on individual patient needs. Both guidelines recommend C1INH as first-line/preferred therapy for LTP in a range of patient types including adults, children/adolescents, and pregnant/lactating patients. The HAEA also recommends the kallikrein inhibitor, lanadelumab, as a first-line option for LTP. HAE pathway-specific agents for LTP have not been associated with notable safety concerns. DISCUSSION: Plasma-derived C1INH has been available for 40+ years in Europe and impacts multiple targets within the HAE pathway. C1INH has been used for on-demand treatment and LTP. A subcutaneous formulation of plasma-derived C1INH is approved for LTP and produces functional C1INH activity levels consistently above the threshold needed for protection from HAE attacks. Other pathway-specific options for LTP include the plasma kallikrein inhibitors, lanadelumab-flyo and berotralstat, approved for adults and pediatric patients aged ≥12 years. C1INH is approved for adults and pediatric patients aged ≥6 years. CONCLUSION: Assessing the need for LTP is vital in the ongoing dialogue between clinicians and patients, as both disease-related factors and patient preferences may change over time. Among available options for LTP, plasma-derived C1INH is the broadly recommended first-line option for LTP in patients with HAE, including pregnant/lactating women and pediatric patients (≥6 years).

Hereditary angioedema: Comprehensive management plans and patient support.

Hereditary angioedema (HAE) is a rare disease. Regardless, patients with HAE have access to multiple state-of-the-art medications available for on-demand use and prevention that reduce the frequency and burden of HAE attacks. These treatments have greatly reduced the burden of disease and helped patients achieve improved quality of life. However, with greater numbers of therapeutic options, HAE care has become more complex. In this review, we addressed essential elements of an individualized comprehensive management plan for a patient with HAE. We focused on access to an expert physician, ongoing patient education, access to effective treatment options, coordination of care and management of treatment logistics, ongoing monitoring of attacks and treatments, and other resources for patient support. This plan will need to be communicated with the patient and other care providers, especially during emergent conditions, and accommodate the patient's lifestyle with consideration for work, school, travel, etc. Periodically, the physician and the patient will need to review information about attacks, triggers, and treatments to identify areas for improvement and update the plan.

Unique Lipid Signatures of Extracellular Vesicles from the Airways of Asthmatics.

Asthma is a chronic inflammatory disease process involving the conductive airways of the human lung. The dysregulated inflammatory response in this disease process may involve multiple cell-cell interactions mediated by signaling molecules, including lipid mediators. Extracellular vesicles (EVs) are lipid membrane particles that are now recognized as critical mediators of cell-cell communication. Here, we compared the lipid composition and presence of specific lipid mediators in airway EVs purified from the bronchoalveolar lavage (BAL) fluid of healthy controls and asthmatic subjects with and without second-hand smoke (SHS) exposure. Airway exosome concentrations were increased in asthmatics, and correlated with blood eosinophilia and serum IgE levels. Frequencies of HLA-DR+ and CD54+ exosomes were also significantly higher in asthmatics. Lipidomics analysis revealed that phosphatidylglycerol, ceramide-phosphates, and ceramides were significantly reduced in exosomes from asthmatics compared to the non-exposed control groups. Sphingomyelin 34:1 was more abundant in exosomes of SHS-exposed asthmatics compared to healthy controls. Our results suggest that chronic airway inflammation may be driven by alterations in the composition of lipid mediators within airway EVs of human subjects with asthma.

Optimization of recombinant adeno-associated viral vectors for human beta-globin gene transfer and transgene expression.

Therapeutic levels of expression of the beta-globin gene have been difficult to achieve with conventional retroviral vectors without the inclusion of DNase I-hypersensitive site (HS2, HS3, and HS4) enhancer elements. We generated recombinant adeno-associated viral (AAV) vectors carrying an antisickling human beta-globin gene under the control of either the beta-globin gene promoter/enhancer or the erythroid cell-specific human parvovirus B19 promoter at map unit 6 (B19p6) without any enhancer, and tested their efficacy in a human erythroid cell line (K-562) and in primary murine hematopoietic progenitor cells (c-kit(+)lin()). We report here that (1) self-complementary AAV serotype 2 (scAAV2)-beta-globin vectors containing only the HS2 enhancer are more efficient than single-stranded AAV (ssAAV2)-beta-globin vectors containing the HS2+HS3+HS4 enhancers; (2) scAAV2-beta-globin vectors recombine with scAAV2-HS2+HS3+HS4 vectors after dual-vector transduction, leading to transgene expression; (3) scAAV2-beta-globin as well as scAAV1-beta-globin vectors containing the B19p6 promoter without the HS2 enhancer element are more efficient than their counterparts containing the HS2 enhancer/beta-globin promoter; and (4) scAAV2-B19p6-beta-globin vectors in K-562 cells, and scAAV1-B19p6-beta-globin vectors in murine c-kit(+)lin() cells, yield efficient expression of the beta-globin protein. Thus, the combined use of scAAV vectors and the parvovirus B19 promoter may lead to expression of therapeutic levels the beta-globin gene in human erythroid cells, which has implications in the use of these vectors in gene therapy of beta-thalassemia and sickle cell disease.

Recombinant self-complementary adeno-associated virus serotype vector-mediated hematopoietic stem cell transduction and lineage-restricted, long-term transgene expression in a murine serial bone marrow transplantation model.

Although conventional recombinant single-stranded adeno-associated virus serotype 2 (ssAAV2) vectors have been shown to efficiently transduce numerous cells and tissues such as brain and muscle, their ability to transduce primary hematopoietic stem cells (HSCs) has been reported to be controversial. We have previously documented that among the ssAAV serotype 1 through 5 vectors, ssAAV1 vectors are more efficient in transducing primary murine HSCs, but that viral second-strand DNA synthesis continues to be a rate-limiting step. In the present studies, we evaluated the transduction efficiency of several novel serotype vectors (AAV1, AAV7, AAV8, and AAV10) and documented efficient transduction of HSCs in a murine serial bone marrow transplantation model. Self-complementary AAV (scAAV) vectors were found to be more efficient than ssAAV vectors, and the use of hematopoietic cell-specific enhancers/promoters, such as the human beta-globin gene DNase I-hypersensitive site 2 enhancer and promoter (HS2-betap) from the beta-globin locus control region (LCR), and the human parvovirus B19 promoter at map unit 6 (B19p6), allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice. The proviral AAV genomes were stably integrated into progenitor cell chromosomal DNA, and did not lead to any overt hematological abnormalities in mice. These studies demonstrate the feasibility of the use of novel scAAV vectors for achieving high-efficiency transduction of HSCs as well as erythroid lineage-restricted expression of a therapeutic gene for the potential gene therapy of beta-thalassemia and sickle cell disease.

Stable integration of recombinant adeno-associated virus vector genomes after transduction of murine hematopoietic stem cells.

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

A combined therapeutic approach for pyruvate dehydrogenase deficiency using self-complementary adeno-associated virus serotype-specific vectors and dichloroacetate.

We determined the ability of self-complementary adeno-associated virus (scAAV) vectors to deliver and express the pyruvate dehydrogenase E1alpha subunit gene (PDHA1) in primary cultures of skin fibroblasts from 3 patients with defined mutations in PHDA1 and 3 healthy subjects. Cells were transduced with scAAV vectors containing the cytomegalovirus promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a vector:cell ratio of 200. Transgene expression was measured 72h later. The transduction efficiency of scAAV2 and scAAV6 vectors was 3- to 5-fold higher than that of the other serotypes, which were subsequently used to transduce fibroblasts with wild-type PDHA1 cDNA under the control of the chicken beta-action (CBA) promoter at a vector:cell ratio of 1000. Total PDH-specific activity and E1alpha protein expression were determined 10 days post-transduction. Both vectors increased E1alpha expression 40-60% in both control and patient cells, and increased PDH activity in two patient cell lines. We also used dichloroacetate (DCA) to maximally activate PDH through dephosphorylation of E1alpha. Exposure for 24h to 5mM DCA increased PDH activity in non-transduced control (mean 37% increase) and PDH deficient (mean 44% increase) cells. Exposure of transduced patient fibroblasts to DCA increased PDH activity up to 90% of the activity measured in untreated control cells. DCA also increased expression of E1alpha protein and, to variable extents, that of other components of the PDH complex in both non-transduced and transduced cells. These data suggest that a combined gene delivery and pharmacological approach may hold promise for the treatment of PDH deficiency.

Adeno-associated virus-mediated gene transfer in hematopoietic stem/ progenitor cells as a therapeutic tool.

Hematopoietic stem cells (HSCs) have unique properties of self-renewal, differentiation and proliferation. HSCs are easily accessible, and can be readily delivered back to patients by autologous transplantation, which renders them as attractive targets for ex vivo gene therapy. The adeno-associated virus (AAV) vectors have to date not been associated with any malignant disease, and have gained attention as a potentially safer alternative to the more commonly used retroviral vectors for HSC gene therapy. Although conflicting data exist with regard to HSC transduction by AAV vectors, in this review, we provide an overview of AAV-mediated HSC gene transfer - obstacles as well strategies to improve the transduction efficiency - and the potential use of AAV vectors for gene therapy of human diseases involving HSCs.

Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5.

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.

Impaired nuclear transport and uncoating limit recombinant adeno-associated virus 2 vector-mediated transduction of primary murine hematopoietic cells.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.