High-Throughput HLA-Typing: on Raw, Unpurified Cord Blood Samples

描述（由申请人提供）：摘要。未实现的机会：准确、简单、廉价、高通量的HLA分型，作为配套遗传学检测，支持国家规模的干细胞治疗计划。已经清楚的是，出生时容易从脐带（UCB）获得的50- 100 ml血液可以成为多能间充质干细胞的丰富的、相对便宜的来源，用于治疗淋巴癌、地中海贫血，并且最近作为更大范围的潜在干细胞疗法的潜在来源。随着正在进行的研究揭示了脐带血干细胞治疗的全部托盘（糖尿病，神经系统疾病，心血管疾病，创伤等），将UCB干细胞的收集和处理扩大到接近出生队列的水平是可行的（在美国每天12，000）。然而，为了支持即使是10%的应计率，也需要大幅增强UCB样本处理和样本分析。在这个R21中，我们专注于这种异基因UCB干细胞治疗所需的伴随基因检测的规模扩大：处理的UCB干细胞样本和从中受益的患者人群的HLA分型。解决方案：廉价的微阵列，用于对新鲜脐带血中的原始干细胞进行HLA分型，或者更好地，从UCB中收集并在古特里卡上干燥运输。我们将优化和验证一套简单而廉价的用于HLA分型的微阵列技术，我们已经在初步结果中展示了这套技术，用于处理原始的、完全未纯化的成人血液和在古特里卡上收集和运输的匹配原始样本。对R21特别重要的是获得来自NHLBI资助的COBLT研究的高度验证的（原始）UCB标本，这是开创性的UCB标本（见支持信）。通过COBLT，使用“金标准”基因分型技术，已经在这些UCB样本上获得了高分辨率的HLA分型。在这个R21中，我们将重点关注HLA-A，HLA-B和DRB 1基因，这些基因与UCB治疗结果和HLA-C和DQB 1的相关性最强，有初步数据表明其在优化UCBT结果中的作用。特别重要的是我们的证明，这种“原始样本HLA分型”可以优化，以工作在干燥的富集UCB样本，收集和运输的古特里卡，通过一种新的回收方法，我们“只是加水”，以产生一个样品准备分析。验证这套耦合微阵列技术，支持基于UCB的干细胞治疗，将由4个特定目标支持：SA 1。1、2岁。生产低成本的HLA-Chips [HLA-A，HLA-B，HLA-C，DRB 1 & DQB 1]。 SA 2.一岁。使用480份原始富集UCB样本优化HLA分型。 SA 3.两岁优化480份原始富集UCB标本的HLA分型：在古特里卡上干燥。 SA 4.两岁对新鲜和干燥的UCB样品进行自动化HLA分型。与后续R33的关系。在本R21中，我们将证明5 μ L富集的UCB可以移液到古特里卡中，邮寄干燥，然后通过“只需加水”进料到我们的高通量HLA分型工作流程中。在后续的R33中，我们将利用R21验证作为与NHLBI附属组织（如世界骨髓捐献者协会脐带血工作组）进行更大规模前瞻性验证的基础。虽然雄心勃勃，但这个想法是可行的：考虑到这个R21团队（BK）中的一个曾是新千年发展目标国际委员会主席。在该R33中，当前用于采集、处理和HLA分型的SOP将继续保持不变：但同时，将在处理点将5 uL的每个富集的UCB样本转移到古特里卡中，匿名，在R21中开发的系统的放大版本上，在国际上邮寄到GenUSA进行高通量HLA分型。然后将该“原始古特里卡”HLA分型在准确性和成本方面与通过纯化DNA上的HLA分型的当前方法获得的HLA分型进行比较。

DESCRIPTION (provided by applicant): Abstract. The Unmet Opportunity: Accurate, simple, inexpensive, high throughput HLA-Typing, as the Companion Genetic Test to support a national-scale program of stem cell therapy. It has become clear that the 50-100mls of blood, readily obtained at birth from the umbilical cord (UCB) can become an abundant, relatively-inexpensive source of pluripotent mesenchymal stem cells to be used in the treatment of lymphoid cancers, the thalassemias and more recently, as the potential source for a much larger range of potential stem cell therapies. As ongoing research reveals the full pallet of cord blood stem cell therapies (diabetes, neurological disease, cardiovascular disease, trauma, etc) it could be feasible to scale-up the collection and processing of UCB stem cells to a level that would approximate the birth cohort (@12,000/day in the US). However, to support even 10% of that accrual rate, substantial enhancement of UCB sample processing and sample analysis will be required. In this R21, we focus on the scale-up of the Companion Genetic Test required for such allogenic UCB stem cell therapy: HLA-Typing of both the processed UCB stem cell samples and the patient population who would benefit from them. The Solution: Inexpensive Microarrays for HLA-Typing of raw stem cells from fresh umbilical cord blood, or better, from UCB collected and shipped dry on Guthrie Cards. We will optimize & validate a simple & inexpensive suite of microarray technologies for HLA-Typing, which we have already shown in Preliminary Results, to work on raw, completely-unpurified adult blood and on matched raw samples collected & shipped dry on Guthrie Cards. Of special importance to this R21, is access to highly-validated (raw) UCB specimens from the NHLBI-funded COBLT study, the seminal UCB c0nsortium (see Letter of Support). High resolution HLA-typing has already been obtained, by COBLT, on those UCB samples, using "gold-standard" genotyping technologies. In this R21, we will focus on HLA-A, HLA-B and DRB1 genes, which are known to have the strongest associations with UCB therapeutic outcome and HLA-C & DQB1 for which there are preliminary data to suggest a role in optimized UCBT outcomes. Of special logistical importance will be our demonstration that such "Raw Sample HLA-Typing" can be optimized to work on dried enriched UCB samples, collected and shipped on Guthrie cards, via a novel recovery approach where we "just add water" to produce a sample ready for analysis. Validation of this set of coupled microarray technologies, in support of UCB-based stem cell therapy, will be supported by 4 Specific Aims: SA1. Yrs 1,2. Manufacture low-cost HLA-Chips [HLA-A, HLA-B, HLA-C, DRB1 & DQB1]. SA2. Yr-1. Optimize HLA-Typing with 480 raw, enriched UCB samples. SA3. Yr-2. Optimize HLA-Typing on 480 raw, enriched UCB specimens: dry on Guthrie Cards. SA4. Yr-2. Automated, HLA-Typing of raw, enriched UCB samples: fresh & dried. Relation to a Follow-on R33. In this R21, we will demonstrate that 5uL of enriched UCB can be pipetted to a Guthrie Card, mailed dry, then fed-into our high throughput HLA-Typing workflow by "just adding water". In a follow-on R33, we will leverage this R21 validation as the basis for a much larger, prospective validation with a NHLBI-affiliated group such as the Cord Blood Working Group of the World Marrow Donor Association. Although ambitious, that idea is feasible: given that one of this R21 Team (BK) was previously Chairman of the International Committee of The NMDP. In that R33, the current SOPs for collection, processing & HLA-Typing would continue unaltered: but, in parallel, 5uL of each enriched UCB sample would be transferred to a Guthrie Card at point of processing, anonymized, mailed internationally to GenUSA for high throughput HLA-Typing, on a scaled-up version of the system developed in the R21. That "Raw Guthrie Card" HLA-Typing would then be compared, in terms of accuracy & cost, to that obtained by current methods of HLA-Typing on purified DNA.