The challenges for in vivo editing of the hematologic system are currently even more difficult than those outlined for ex vivo editing (which is built on the established HSCT and HCT platforms). These challenges fall into two broad categories: (1) delivery and (2) immunogenicity.

Delivery

In vivo genome editing requires the development of a novel vector that would specifically target the cell of interest (whether HSCs or T cells or other blood cell) while not being taken up by the trillions of other cells (red blood cells, platelets, endothelial cells…) that the target cells live and coexist with in close proximity. In contrast to ex vivo editing where the target cell can be purified way from the competitor cells before editing, in an in vivo setting that would not possible. Furthermore, as discussed earlier, to achieve high frequencies of genome editing, whether through indels or HDR, requires a sufficient quantity of the genome editing reagent to enter the cell. A little gets only a little. Thus the new vector would not only have to selectively deliver to the target cell of interest but would also have to deliver the editing reagents in sufficient quantities to achieve therapeutic frequencies of editing. Finally, it should be noted that in terms of HDR, which requires cells to be cycling to be efficient, most HSCs are quiescent and so to harness HDR in vivo would require novel ways to put HSCs into cycle without compromising their long-term hematopoietic stem cell function.

Immunogenicity

The human immune system has been one of the barriers to safe and effective in vivo gene therapy, and one would anticipate the same to be true for in vivo gene editing. The first barrier is to avoid activating the intracellular innate immune response (type I interferon response) in all cells to which the novel vector delivered its cargo in vivo. The second barrier is to design a vector that does not profoundly activate the cellular innate immune response, such as macrophages and the reticuloendothelial system. The third barrier is the adaptive immune response to the genome editing reagents and vector. All of the nuclease systems involve parts that are foreign. For CRISPR/Cas9, the Cas9 protein has been demonstrated to generate an adaptive immune response, even in naïve animals.[1,2] Therefore, for in vivo editing, one would need enough nuclease to get therapeutic levels of editing but have it disappear before the adaptive immune response recognized the foreign protein and eliminated the cells still expressing it (like a virus). Moreover, most healthy adults already have a preexisting immune response to both Staphylococcus aureus and S. pyogenes Cas9.[3,6] For in vivo AAV gene therapy, patients who have even very low titers of preexisting antibodies to the AAV serotype are excluded from treatment because the preexisting immunity diminishes efficacy and might create systemic inflammation, toxicity, and death.

Proponents of in vivo editing also argue that it would be cheaper than an ex vivo process, but this assumption has not been pressure tested. A simple counter example is that the current most expensive drug in the world is an in vivo AAV-based gene therapy for spinal muscular atrophy, not an ex vivo HSPC gene therapy. In addition, the amount of AAV (the current best vector) for systemic in vivo delivery of nucleic acids needed for one patient for in vivo editing would be sufficient for thousands, if not tens of thousands, of patients using an ex vivo approach.

Nonetheless, ex vivo editing of HSCs at levels that might cure hematologic diseases was also considered an unrealistic dream not even two decades ago, so the creativity, ambition, and motivation of a large number of researchers might, over the next decades, discover solutions to the known challenges described earlier. We can anticipate that solving currently unknown barriers to in vivo editing will have to, and will, occur.

References

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[1] Nelson CE, et al. Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nat Med. 2019;25(3):427–432.

[2] Li A, et al. AAV-CRISPR gene editing is negated by pre-existing immunity to Cas9. Mol Ther. 2020;28(6):1432–1441.

[3] Charlesworth CT, et al. Identification of pre-existing adaptive immunity to Cas9 proteins in humans. BioRxiv. 2018.

[4] Wagner DL, et al. High prevalence of S. pyogenes Cas9-specific T cell sensitization within the adult human population – A balanced effector/ regulatory T cell response. BioRxiv. 2018.

[5] Simhadri VL, et al. Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4(+) T-cells. Nat Commun. 2021;12(1):5090.

[6] Simhadri VL, et al. Prevalence of pre-existing antibodies to CRISPR associated nuclease Cas9 in the USA population. Mol Ther Methods Clin Dev. 2018;10:105–112.

اخر الاخبار

اخبار العتبة العباسية المقدسة

أهالي كربلاء يحيون اليوم السابع لذكرى شهادة الإمام الحسين (عليه السلام)

مشروع أمير القراء الوطني يشهد مشاركة أكثر من 100 طالبٍ في مرحلته الثالثة

قسم الشؤون الفكرية يصدر دليلًا يوثّق القصص الفائزة في مسابقة فتوى الدفاع المقدسة

قسم الشؤون الفكرية يعلن عن توفير عددٍ من المكتبات الإلكترونية المتخصّصة للمؤسّسات الدينية والمكتبات العامّة

المزيد

الآخبار الصحية

نصائح عملية للحد من التعرق المفرط في الصيف

أطباء يحذرون: مكمل غذائي شائع قد يدمر الجهاز العصبي

تحذير طبي من تأثير جانبي "غير متوقع" لحقن التخسيس

حمامات الثلج قد تكون خطيرة وأحيانا مميتة.. ما السبب؟

المزيد

الآخبار التكنلوجية

أكبر جدارية شمسية في العالم تدخل موسوعة غينيس.. مشروع ضخم

تسارع دوران الأرض يثير القلق.. وتحذيرات من "كوارث محتملة" ؟

"سوذبيز" تطرح أكبر قطعة من المريخ على الأرض في مزاد علني

أوبن أيه آي تعتزم إطلاق متصفح إنترنت يعمل بالذكاء الاصطناعي

المزيد

مواضيع ذات صلة

Heritable Genome Editing

EX Vivo Manufacturing of Therapeutic Cells

Using Engineered Nucleases to Initiate the Genome Editing Process

Base Editing

Double-stranded DNA breaks: the basis of nuclease-based genome editing

Plaque and Colony Hybridization for Clone Identification

Cloning from RNA

Plasmid Vectors

كلونة النعجة دولي Cloning Dolly the sheep

ادخال الجينات في الخلايا البدائية النواة

أدوات نقل الـ DNA نواقل الكلونة (cloning vehicles)

أدوات كلونة الجين المتعاقبة