Gene Delivery to Human Limbal Stem Cells Using Viral Vectors

Song, Liujiang; Song, Zhenwei; Fry, Nathaniel J; Conatser, Laura; Llanga, Telmo; Mei, Hua; Kafri, Tal; Hirsch, Matthew

doi:10.1089/hum.2019.071

Cited by 10 publications

(6 citation statements)

References 34 publications

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“…However, to date, there is only one study that investigated the transduction efficiency of human LSCs using AAVs. Song et al added eight different self-complementary AAV serotypes (AAVs 1–9) harboring GFP reporter to human limbal epithelial cells that were isolated in vitro and observed that AAV 6 demonstrated the highest GFP transgene expression at 24 h post AAV-exposure, followed by AAV 4 20 . Notably, the authors also performed intrastromal injection of AAV 6 in central ex vivo corneas to test the transduction efficiency of LSCs, a technique which has been shown to be feasible in mice corneas 21 .…”

Section: Discussionmentioning

confidence: 99%

In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study

Son

Jun

Foster

et al. 2022

Sci Rep

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This study aimed to evaluate the efficacy of in situ adeno-associated virus (AAV)-mediated gene delivery into the human corneal limbal region via targeted sub-limbal injection technique. Human cadaveric corneal tissues were fixed on an artificial anterior chamber. Feasibility of sub-limbal injection technique was tested using trypan blue and black India ink. An enhanced green fluorescent protein (eGFP) encoding AAV DJ was injected into sub-limbal region. After AAV injection, corneal tissues were incubated in air-lift culture and prepared for immunohistochemical analysis. Cell survivial and expression of eGFP, stem cell markers (p63α and cytokeratin 19 (KRT19)), and differentiation marker cytokeratin 3 (KRT3) were evaluated using confocal microscopy. Both trypan blue and black India ink stained and were retained sub-limbally establishing specificity of the injection technique. Immunohistochemical analysis of corneas injected with AAV DJ-eGFP indicated that AAV-transduced cells in the limbal region co-express eGFP, p63α, and KRT19 and that these transduced cells were capable of differentiating to KRT3 postitive corneal epithelial cells. Our sub-limbal injection technique can target cells in the human limbus in a reproducible and efficient manner. Thus, we demonstrate that in situ injection of corneal limbus may provide a feasible mode of genetic therapy for corneal disorders with an epithelial etiology.

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Section: Discussionmentioning

confidence: 99%

In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study

Son

Jun

Foster

et al. 2022

Sci Rep

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“…Furthermore, our results are in line with observations made in other studies showing that the genetic modification of LSCs does not have a detrimental effect in cell proliferation or stemness. Even though in our approach we did not evaluate stratification of epithelial cells after transduction, it has been described that gene delivery using lentiviral particles does not alter their clonogenic capacity (54)(55)(56).…”

Section: Discussionmentioning

confidence: 99%

Genetic Modification of Limbal Stem Cells to Decrease Allogeneic Immune Responses

Valdivia¹,

Bertolin

Breda

et al. 2021

Front. Immunol.

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Limbal stem cell (LSC) transplantation is the only efficient treatment for patients affected by LSC deficiency (LSCD). Allogeneic LSC transplantation is one of the most successful alternative for patients with bilateral LSCD. Nevertheless, the high variability of the human leukocyte antigens (HLA) remains a relevant obstacle to long-term allogeneic graft survival. This study characterized the immunologic properties of LSCs and proposed a genetic engineering strategy to reduce the immunogenicity of LSCs and of their derivatives. Hence, LSC HLA expression was silenced using lentiviral vectors encoding for short hairpin (sh) RNAs targeting β2-microglobulin (β2M) or class II major histocompatibility complex transactivator (CIITA) to silence HLA class I and II respectively. Beside the constitutive expression of HLA class I, LSCs showed the capability to upregulate HLA class II expression under inflammatory conditions. Furthermore, LSCs demonstrated the capability to induce T-cell mediated immune responses. LSCs phenotypical and functional characteristics are not disturbed after genetic modification. However, HLA silenced LSC showed to prevent T cell activation, proliferation and cytotoxicity in comparison to fully HLA-expressing LSCs. Additionally; HLA-silenced LSCs were protected against antibody-mediated cellular-dependent cytotoxicity. Our data is a proof-of-concept of the feasibility to generate low immunogenic human LSCs without affecting their typical features. The use of low immunogenic LSCs may support for long-term survival of LSCs and their derivatives after allogeneic transplantation.

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“…Although the transduction efficiency of rAAV in different stem cells including embryonic stem cells (ESCs), hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs) has been widely studied [ 92 ], to the best of our knowledge, there is no comparative transduction study of CSCs in vitro or in vivo. Although AAV6 capsids with site-specific modifications and CD34+ HSC-derived AAV variants are reported to have higher tropism to blood stem cells and support stable and efficient gene transfer [ 93 ], most other AAV serotypes may not productively transduce stem cells efficiently [ 94 , 95 ]. Unfortunately, cancer or CSC-derived AAV capsids have not yet been reported to date, consistent with reports of low to no germline transmission of AAV and an inverse relationship of wtAAV integrants in cancerous versus non-transformed cells.…”

Section: Targeting Cancer Stem Cells With Aavmentioning

confidence: 99%

“…Similarly, Hirsch et al found that ESCs are particularly sensitive to AAV-induced cell death via a p53-dependent apoptotic response that is elicited by a telomeric sequence within the AAV ITR [ 59 , 92 ]. Reports of toxicity to other types of stem-like cells have shown mixed results, alluding to potentially differential effects of AAV toxicity depending on the species, type of stem cell, and AAV serotype and have been thoroughly discussed in a previous review [ 92 , 95 , 99 , 100 , 101 , 102 ]. When toxicity is observed, it is mostly thought to be induced through TLR activation via DNA sensors, especially the palindromic ITR hairpin DNA sequences [ 103 ].…”

Section: Targeting Cancer Stem Cells With Aavmentioning

confidence: 99%

Harnessing the Natural Biology of Adeno-Associated Virus to Enhance the Efficacy of Cancer Gene Therapy

Bower

Song

Bastola

et al. 2021

Viruses

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Adeno-associated virus (AAV) was first characterized as small “defective” contaminant particles in a simian adenovirus preparation in 1965. Since then, a recombinant platform of AAV (rAAV) has become one of the leading candidates for gene therapy applications resulting in two FDA-approved treatments for rare monogenic diseases and many more currently in various phases of the pharmaceutical development pipeline. Herein, we summarize rAAV approaches for the treatment of diverse types of cancers and highlight the natural anti-oncogenic effects of wild-type AAV (wtAAV), including interactions with the cellular host machinery, that are of relevance to enhance current treatment strategies for cancer.

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Gene Delivery to Human Limbal Stem Cells Using Viral Vectors

Cited by 10 publications

References 34 publications

In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study

In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study

Genetic Modification of Limbal Stem Cells to Decrease Allogeneic Immune Responses

Harnessing the Natural Biology of Adeno-Associated Virus to Enhance the Efficacy of Cancer Gene Therapy

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