General description of the study

This is a prospective, multicenter, expanded access interventional study of subjects recovered from COVID-19 pneumonia to assess their response to intravenous administration of adipose-derived autologous SVF.

Primary objective

The purpose of this study was to evaluate the safety of single intravenous injections of autologous adipose-derived SVF produced using the GID SVF-2 device system for the treatment of secondary respiratory distress associated with COVID-19.

Secondary objective

To evaluate the efficacy of the initial treatment with SVF IV.

The recent outbreak of Coronavirus 2 (SARS-CoV-2) has spread rapidly throughout the world, resulting in a global pandemic with devastating socioeconomic consequences. After being declared a public health emergency by the World Health Organization (WHO), there is an urgent need to develop effective therapeutic strategies for critically ill COVID-19 patients. This new virus strain causes a complex disease with a wide range of presentations, from mild symptoms to multi-organ failure. A common feature of severe cases is the pathologically complex "cytokine storm" that presents as an excessive immune response with rapid progression of disease and high mortality. In particular, the severe outcomes of SARS CoV-2 are associated with elevated C-reactive protein and Interleukin-6 in the lungs. COVID-19 infection can rapidly decompensate into severe respiratory failure requiring intubation and mechanical ventilation. The need for mechanical ventilation portends a poor prognosis, with a reported mortality rate of up to 88%.

Survivors of COVID-19 pneumonia face sequelae of their disease that affect multiple organ systems. In particular, a significant number present with ongoing problems of breathlessness and reduced oxygenation, turning previously healthy patients into virus-induced pulmonary cripples. The mechanism for this is the intense scarring and destruction of the microcirculation found in the lungs of COVID-19 survivors. There is an urgent need for the development of treatment protocols that are capable of reducing the degree of pulmonary fibrosis and promoting local angiogenesis to better support injured alveoli.

In recent decades, mesenchymal stromal cells (MSCs) have emerged as a potential therapeutic agent for cell-based therapies due to the beneficial effects on immunomodulation and tissue repair/regeneration. These cells possess properties unique self-renewal and capacity to differentiate into multiple lineages. MSCs are found in small numbers in bone marrow (BMSC) and umbilical cord tissue. MSCs are also found in adipose tissue (referred to as ASCs) where they exist as part of a multicellular population, the stromal vascular fraction (SVF). ASC populations are 500-1000 more abundant than their bone marrow counterparts.

Adipose tissue provides a source of Stromal Vascular Fraction (SVF) that can be isolated and transplanted to the patient during the same surgical procedure, at the point of care. SVF is a heterogeneous mixture of stromal progenitor cells, pericytes, endothelial precursor cells, and macrophages. Acting collectively, SVF has been shown to possess broad anti-inflammatory and regenerative properties. SVF has been shown to be safe after IV administration and has shown some promising results in restoring respiratory function in patients with severe lung disorders. Based on public analysis of single cell RNA sequencing (scRNA-seq) data, SVF demonstrates the absence of ACE2 expression, indicating its potential as a resistant phenotype to SARS-CoV-2 infection. In Taken together, IV administration of adipose- derived SVF is presented as a novel treatment approach to improve the clinical outcome of respiratory-compromised COVID-19 patients.

The clinical impact of SVF for COVID-19 is based on 5 mechanisms of action. These have been widely documented (see attached publications and bibliography).

Anti-inflammatory Immunomodulation, especially T-regs Antifibrosis. Matrix metalloproteinases and liver growth factor. Support for regenerative cell populations in situ. Lung asthma studies Angiogenesis under ischemic conditions, based on the release of VEGF.

Therapy with SVF cells from adipose tissue is advantageous as large numbers of cells can be removed from small volumes (30-90 cc) by a minimally invasive liposuction procedure.

Indication for expanded access

This is an expanded access study to treat a small group of subjects with pulmonary sequelae after recovery from COVID-19 pneumonia of autologous adipose-derived SVF administered using as single intravenous injection.

Objectives of clinical research

Main objective

To assess the safety of a single injection of autologous adipose-derived SVF produced with the GID SVF-2 device for the treatment of respiratory distress.

associated with COVID-19.

Secondary objective

To assess efficacy, by (1) maintaining SaO2 saturation ≥ at the existing level on noninvasive oxygen support, (2) achieving a reduction in the level of oxygen support required to maintain SaO2 ≥ 92, using intravenous injection of autologous adipose derived SVF produced using the GID SVF-2 device system for the treatment of respiratory distress associated with COVID-19.

Expected duration of the clinical investigation

Follow-up controls at 3, 6, 9, and 12 months. The total duration of the study is 1 year.

Clinical Protocol

Study design

General study design

This is a prospective, multicenter, expanded access interventional study of subjects with COVID-19. Forty (40) subjects with confirmed COVID-19 and SaO2 ≤ 92 were treated. Subjects received an intravenous injection of autologous adipose-derived SVF. Subjects will be followed for 6 weeks.

Study procedures

Detection procedures

The initial evaluation was done at the local Centro de Salud. Subjects were then referred to HEODRA or HECAM for confirmatory diagnosis and additional tests.

Concomitant medications

All concomitant medications considered Standard of Care are accepted. A concomitant medication case report form will be completed at each subject follow-up visit.

Summary of study treatment

40 non-randomized patients will be treated with autologous SVF. Minimum dosage: 45x106 ± 5x106 cells Treatment plan: a single intervention

Follow-up Serum samples (20 cc) - inflammation factors: 1 month, 3 months, 6 months PFTs + DLco: preop, 1 month, 3 months, 6 months weeks, and 12 months CT: preop, 3 months, 6 months, 12 months SF-36 quality of life questionnaire SF-36 (Medical Outcomes Trust): pre-op, 12 months SGRQ-C respiratory questionnaire SGRQ-C (St. George's University): pre-op, 12 months

The subject's adipose tissue will be acquired by liposuction of the abdomen or flanks and placed directly into the GID SVF-2 device. The harvested adipose tissue will be enzymatically digested in the same GID SVF-2 device using the GIDZyme-2-70 enzyme and centrifuged in the same GID SVF-2 device to concentrate the SVF cells. SVF cells will be removed and an active treatment dose of 45 x 106 (±5 x 106) SVF cells will be injected into a 100 ml IV bag containing LR. Fluids will be given through an IV catheter through a blood filter over 10 minutes.

Dosage

Dose preparation

Using the LunaStem® Nucleocounter Cell Concentration and Dilution Factor 100 calculate the volume needed using the following equation and transfer that amount of resuspension to a 10 mL syringe.

#ml = dose = 40 x 106 (+/- 5x106)

Dose administration The way to administer SVF for vascular use is the same whether it is intravenous (IV) or intravenous (IA).

The treatment was administered intravenously using an intravenous catheter with a blood filter.

Add the dose to a 100 ml bag of LR that has been warmed to 37°C and mix well. Administer the 100 ml over 10 minutes.

Adherence to treatment

Each subject had time to read the consent form and ask questions about the study before signing the informed consent. Subjects should contact the physician or staff if participant have any concerns during the study. It will be emphasized that the subject must comply with the protocol and be honest about her symptoms.

Withdrawal of subjects for non-compliance

Subjects may be terminated from the study at the discretion of the principal investigator only for reasons related to study examinations that would jeopardize the subject's health and/or welfare if participants were to continue in the study. Subjects may voluntarily withdraw from the study at any time without prejudice.

• Subjects withdrawn will not be replaced if participant have received study treatment.

Schedule of study visits

The initial evaluation and informed consent took place in the hospitals. Prior to treatment, subjects were evaluated to determine if participant meet the inclusion/exclusion criteria. If not already provided, demographics, medical history, concomitant medications, SaO2, and arterial blood gases was collected. The subject were enrolled if participant meet all the eligibility criteria and have signed the Informed Consent.

The time between enrollment and treatment not exceeded 48 hours.

On the day of treatment, each subject was reassessed for inclusion in the study.

Inclusion Criteria:

Patients were identified at Managua and Leon, Nicaragua, community health centers and screened for eligibility by study physicians.

• Forty PCR-confirmed COVID-19 patients
• Persistent pulmonary complaints of dyspnea for at least 2 months after hospital discharge.
• Age 18 - 85 years.
• Male or Female.
• A body mass index of > 22.
• Forced vital capacity (FVC) > 40% predicted and < 70% predicted
• Diffusing lung capacity of the lungs for carbon monoxide (DLCO) > 20% predicted and < 70% predicted.

Exclusion Criteria:

• Use of home oxygen
• History of pulmonary malignancy.
• Immunosuppressive drug treatment
• History of prior cardiac disease with an ejection fraction of ≤30%
• Diabetes
• Pregnancy or plans to conceive during the study period.
• Participation in another clinical study.